Diverse CD8 Research Articles

Oligoclonal TRBV gene usage among CD8+ T cells in monoclonal B lymphocytosis and CLL

Oligoclonal <i>TRBV</i> gene usage among CD8⁺ T cells in monoclonal B lymphocytosis and CLL

Clonal Focusing of Epitope-Specific CD8+T Lymphocytes in Rhesus Monkeys following Vaccination and Simian-Human Immunodeficiency Virus Challenge

To afford the greatest possible immune protection, candidate human immunodeficiency virus (HIV) vaccines must generate diverse and long-lasting CD8(+) T lymphocyte responses. In the present study, we evaluate T-cell receptor Vbeta (variable region beta) gene usage and a CDR3 (complementarity-determining region 3) sequence to assess the clonality of epitope-specific CD8(+) T lymphocytes generated in rhesus monkeys following vaccination and simian-human immunodeficiency virus (SHIV) challenge. We found that vaccine-elicited epitope-specific CD8(+) T lymphocytes have a clonal diversity comparable to those cells generated in response to SHIV infection. Moreover, we show that the clonal diversity of vaccine-elicited CD8(+) T-lymphocyte responses is dictated by the epitope sequence and is not affected by the mode of antigen delivery to the immune system. Clonal CD8(+) T-lymphocyte populations persisted following boosting with different vectors, and these clonal cell populations could be detected for as long as 4 years after SHIV challenge. Finally, we show that the breadth of these epitope-specific T lymphocytes transiently focuses in response to intense SHIV replication. These observations demonstrate the importance of the initial immune response to SHIV, induced by vaccination or generated during primary infection, in determining the clonal diversity of cell-mediated immune responses and highlight the focusing of this clonal diversity in the setting of high viral loads. Circumventing this restricted CD8(+) T-lymphocyte clonal diversity may present a significant challenge in the development of an effective HIV vaccine strategy.

Cutting Edge: Size and Diversity of CD4+CD25high Foxp3+ Regulatory T Cell Repertoire in Humans: Evidence for Similarities and Partial Overlapping with CD4+CD25− T Cells

Both differentiation and function of CD4+CD25(high) naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the alphabetaTCR repertoire sizes of human peripheral blood Treg and CD4+CD25- T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted alphabeta TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25- T cells, with a mean estimated size of 3.5 x 10(6) distinct alphabeta TCR vs 4.7 x 10(6) distinct alphabetaTCR for CD4+CD25- T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.

Direct Ex Vivo Analyses of HLA-DR1 Transgenic Mice Reveal an Exceptionally Broad Pattern of Immunodominance in the Primary HLA-DR1-Restricted CD4 T-Cell Response to Influenza Virus Hemagglutinin

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.

DNA immunization using highly conserved murine cytomegalovirus genes encoding homologs of human cytomegalovirus UL54 (DNA polymerase) and UL105 (helicase) elicits strong CD8 T-cell responses and is protective against systemic challenge.

Human cytomegalovirus (HCMV) establishes a lifelong infection with the potential for reinfection or viral transmission even in the presence of strong and diverse CD8 T-lymphocyte responses. This suggests that the CMVs skew the host T-cell response in order to favor viral persistence. In this study, we hypothesized that the essential, nonstructural proteins that are highly conserved among the CMVs may represent a novel class of T-cell targets for vaccine-mediated protection due to their requirements for expression and sequence stability, but that the observed subdominance of these antigens in the CMV-infected host results from the virus limiting the T-cell responses to otherwise-protective specificities. We found that DNA immunization of mice with the murine CMV (MCMV) homologs of HCMV DNA polymerase (M54) or helicase (M105) was protective against virus replication in the spleen following systemic challenge, with the protection level elicited by the M54 DNA being comparable to that of DNA expressing the immunodominant IE1 (pp89). Intracellular gamma interferon staining of CD8 T cells from mice immunized with either the M54 or M105 DNAs showed strong primary responses that recalled rapidly after viral challenge. M54- and M105-specific CD8 T cells were detected after the primary MCMV infection, but their levels were not consistently above the background level. The conserved, essential proteins of the CMVs thus represent a novel class of CD8 T-cell targets that may contribute to a successful HCMV vaccine strategy.

Cytomegalovirus (CMV) IE1‐ and pp65‐Specific CD8+T Cell Responses Broaden over Time after Primary CMV Infection in Infants

Cytomegalovirus (CMV) infection remains a significant cause of morbidity and mortality in young children. We have previously shown that CD8+ T cell responses to CMV pp65 or IE1 protein were readily detectable in children with congenital or postnatal CMV infection. Here, we have further characterized the evolution of the peptide specificity of these responses in 7 infants<6 months of age at the start of the study. Thirteen pp65 and 15 IE1 peptides (median, 5 peptides/infant) were targeted, and most (61%) represented sequences not previously reported. Peptide specificity remained stable or broadened over time despite the clearance of CMV viremia. Loss of peptide recognition was not observed. Responses with the highest functional peptide avidity were not necessarily detected earliest. These data provide additional evidence that young infants can generate diverse CMV-specific CD8+ T cell responses but show that early responses may exhibit relatively focused peptide specificity and lower peptide avidity.

CD8 T cell clonal expansions & aging: A heterogeneous phenomenon with a common outcome

A highly diverse CD8 T cell repertoire is thought to be critical for maintaining appropriate immune defenses against a variety of pathogens. However, in many aged individuals, the diversity of T cell receptors is significantly reduced by the presence of large, monoclonal expansions of CD8 memory T cells. While ongoing research is focused on understanding the molecular alterations in these expansions, one major hurdle to this goal is the apparent heterogeneity of CD8 clonal expansions, which is apparent even in reductionist systems. In this review, we discuss current evidence that CD8 clonal expansions are a heterogeneous phenomenon, and our evolving understanding of what this heterogeneity tells us about CD8 memory T cell homeostasis and how it is altered in aged individuals.

Human virus‐specific CD8+ T cells: diversity specialists

Human viruses elicit functionally highly diverse CD8+ T-cell responses. This variation, which manifests in the memory or latency stage, includes differences in expansion requirements, migratory properties, homeostatic maintenance mechanisms, and constitutive effector properties, and it may reflect specific adaptations of the human immune system to challenges posed by individual pathogens. Longitudinal follow-up of individuals after primary virus infections has revealed how divergent virus-specific CD8+ T cells may develop from the initially expanded virus-specific T-cell effector pool. Recent findings have shown that CD8+ T cells reactive toward latent viruses may depend on other mechanisms for their homeostatic maintenance than T cells specific for cleared viruses. Whereas the latter can respond to the homeostatic cytokine interleukin-7 (IL-7), many persistent virus-specific T cells, recognizing latent herpesviruses such as Epstein-Barr virus and cytomegalovirus (CMV), lack IL-7 receptor alpha (IL-7Ralpha) and depend on viral antigens to persist. Finally, CMV is unique in that it generates a vast pool of resting virus-specific T cells with constitutive cytolytic effector function. The putative role of functionally diverse CD8+ T cells in protective immunity to persistent viruses is discussed in this review.

Genome-Wide Analysis Reveals a Highly Diverse CD8 T Cell Response to Murine Cytomegalovirus

Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2(b)-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.

Contribution of T cell receptor affinity to overall avidity for virus-specific CD8+T cell responses

Prior analysis has characterized the clonal characteristics of effector CD8(+) T cells specific for the prominent influenza A virus nucleoprotein (NP) and acid polymerase (PA) peptides presented by H2D(b). Using a single-cell approach and determination of CDR3beta profiles, a limited, predominantly "public" repertoire was found for CD8(+)D(b)NP(366)(+)Vbeta8.3+ cells, whereas diverse and "private" T cell antigen receptor (TCR)beta clonotypes were typical of the CD8(+)D(b)PA(224)(+)Vbeta7+ response. This single-cell approach has now been used to relate the contributions of particular clonotypes (or affinities) to high-avidity TCRs, as defined by binding under conditions of limiting tetramer availability. At least by the measure of CDR3beta usage, no difference could be found between total and high-avidity populations in the spectrum of TCR-pMHC affinities throughout the limited, and relatively public, CD8(+)D(b)NP(366)(+)Vbeta8.3+ populations. Conversely, the more even (by clone size), diverse, and private CD8(+)D(b)PA(224)(+)Vbeta7+ response was characterized by the clear partitioning of the largest T cell clones in the high-avidity compartment. These results suggest that the relatively constrained CD8(+)D(b)NP(366)(+)Vbeta8.3+ set utilizes a relatively narrow range of affinities, whereas the broader CD8(+)D(b)PA(224)(+)Vbeta7+ response is induced at a range of TCR-pMHC affinities. Thus, whereas TCR sequence (or affinity) appears to contribute substantially to the avidity profile of diverse virus-specific CD8+ populations, other mechanisms may be prominent where the TCR spectrum is more limited.

Perforin and Fas Cytolytic Pathways Coordinately Shape the Selection and Diversity of CD8+-T-Cell Escape Variants of Influenza Virus

Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.

Diverse CD8+ T-Cell Responses to Renal Cell Carcinoma Antigens in Patients Treated with an Autologous Granulocyte-Macrophage Colony-Stimulating Factor Gene-Transduced Renal Tumor Cell Vaccine

A phase I clinical trial with granulocyte-macrophage colony-stimulating factor tumor cell vaccines in patients with metastatic renal cell carcinoma (RCC) showed immune cell infiltration at vaccine sites and delayed-type hypersensitivity (DTH) responses to autologous tumor cells indicative of T-cell immunity. To further characterize RCC T-cell responses and identify relevant RCC-associated antigens, we did a detailed analysis of CD8+ T-cell responses in two vaccinated RCC patients who generated the greatest magnitude of DTH response and also displayed a strong clinical response to vaccination (>90% reduction in metastatic tumor volume). Three separate CD8+ T-cell lines (and subsequent derived clones) derived from patient 24 recognized distinct RCC-associated antigens. One recognized a shared HLA-A*0201-restricted antigen expressed by both renal cancer cells and normal kidney cells. This recognition pattern correlated with a positive DTH test to normal kidney cells despite no evidence of impairment of renal function by the patient's remaining kidney after vaccination. A second line recognized a shared HLA-C7-restricted antigen that was IFN-gamma inducible. A third line recognized a unique HLA-A*0101-restricted RCC antigen derived from a mutated KIAA1440 gene specific to the tumor. In addition, two independent CTL lines and three clones were also generated from patient 26 and they recognized autologous tumor cells restricted through HLA-A*0205, HLA-A/B/C, and HLA-B/C. These results show that paracrine granulocyte-macrophage colony-stimulating factor tumor vaccines may generate a diverse repertoire of tumor-reactive CD8+ T-cell responses and emphasize the importance of polyvalency in the design of cancer immunotherapies.

Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection

AbstractAlthough cytomegalovirus (CMV) expresses proteins that interfere with antigen presentation by class I major histocompatibility complex (MHC) molecules, CD8+ cytotoxic T cells (CTLs) are indispensable for controlling infection and maintaining latency. Here, a cytokine flow cytometry assay that employs fibroblasts infected with a mutant strain of CMV (RV798), which is deleted of the 4 viral genes that are responsible for interfering with class I MHC presentation, was used to examine the frequency and specificity of the CD8+ CTLs to CMV in immunocompetent CMV-seropositive individuals. A large fraction of the CD8+ CTL response was found to be specific for viral antigens expressed during the immediate early and early phases of virus replication and presented by fibroblasts infected with RV798 but not wild-type CMV. These results demonstrate that the inhibition of class I antigen presentation observed in CMV-infected cells in vitro is not sufficient to prevent the induction of a broad repertoire of CD8+ CTLs after natural infection in vivo. Thus, reconstitution of T-cell immunity in immunodeficient patients by cell therapy or by vaccination may need to target multiple viral antigens to completely restore immunologic control of CMV.

Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection.

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14(+) monocytes and CD4(+) T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14(+) monocytes and CD4(+) T cells. Three of the five patients had CD14(+) monocyte populations with significantly more genetic diversity than the CD4(+) T-cell population, while the other two patients had more genetic diversity in CD4(+) T cells. The cellular compartmentalization of HIV-1 between CD14(+) monocytes and CD4(+) T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4(+) T cells and CD14(+) monocytes of two patients. These findings suggest that HIV-1 in CD14(+) monocytes could disseminate and evolve independently from that in CD4(+) T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.

Very diverse CD8 T cell clonotypic responses after virus infections.

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-gamma capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vbeta chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1-2 orders of magnitude than predicted previously.

Presence and localization of T-cell subsets in relation to melanocyte differentiation antigen expression and tumour regression as assessed by immunohistochemistry and molecular analysis of microdissected T cells.

Melanoma-associated antigens may be the driving force behind the lymphocytic infiltrates in melanomas and the occurrence of melanoma regression. To investigate the clinical relevance of melanoma differentiation antigens (MDAs) as T-cell targets, the relationship between the presence and localization of T-cell subsets and the expression of MDAs was studied by immunohistochemistry and the diversity of CD8+ T cells in regressive melanomas was assessed using laser-assisted microdissection. While MDA expression as well as T-cell subset distribution, as assessed by immunohistochemical analysis, was heterogeneous within and between lesions, they were histologically independent phenomena. In four lesions studied in detail by PCR analysis of microdissected T cells, a limited T-cell diversity and evidence for clonally expanded tumour infiltrating lymphocytes were found. However, no major differences in T-cell diversity, as assessed by PCR analysis, between peri-and intra-tumoural areas became apparent, this despite the known clinical significance of the specific localization of a T-cell infiltrate. T cells of clonal origin did not show preferential localization to regressive tumour areas. Moreover, clonally related cells were found in two lesions with a non-brisk infiltrate, while in two lesions with a brisk infiltrate (clinically, a good prognostic factor) no evidence for clonally expanded tumour infiltrating lymphocytes was found. These data support the notion that specific immune reactivity and homing of specific cells to the tumour can occur in melanoma patients. However, they also show that the presence of clonally expanded T cells in the tumour is not necessarily associated with an effective anti-tumour immune response and may, for instance, represent regulatory cells. It appears that the clinical impact of an anti-tumour immune response is largely decided at the tumour site, where micro-environmental conditions dictate the functional state of the T cells. Full understanding of these processes can only be achieved by performing more dynamic analyses of the local host-tumour interactions.

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8 + T cell response in mice and is antigenic for PBMCs from HIV + patients

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8 + T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8 + T cell response measured as production of IFN-γ by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-γ secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV + patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8 + T cell response against conserved viral targets and early expressed regulatory proteins.

Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome).

Chromosome 22q11.2 deletion (del22q11.2) syndrome (DiGeorge syndrome/velocardiofacial syndrome) is a common syndrome typically consisting of congenital heart disease, hypoparathyroidism, developmental delay and immunodeficiency. Although a broad range of immunologic defects have been described in these patients, limited information is currently available on the diversity of the T-cell receptor (TCR) variable beta (BV) chain repertoire. The TCRBV repertoires of nine patients with del22q11.2 syndrome were determined by flow cytometry, fragment size analysis of the third complementarity determining region (CDR3 spectratyping) and sequencing of V(D)J regions. The rate of thymic output and the phenotype and function of peripheral T cells were also studied. Expanded TCRBV families were detected by flow cytometry in both CD4+ and CD8+ T cells. A decreased diversity of TCR repertoires was also demonstrated by CDR3 spectratyping, showing altered CDR3 profiles in the majority of TCRBV families investigated. The oligoclonal nature of abnormal peaks detected by CDR3 spectratyping was confirmed by the sequence analysis of the V(D)J regions. Thymic output, evaluated by measuring TCR rearrangement excision circles (TRECs), was significantly decreased in comparison with age-matched controls. Finally, a significant up-regulation in the percentage, but not in the absolute count, of activated CD4+ T cells (CD95+, CCR5+, HLA-DR+), IFN-gamma - and IL-2-expressing T cells was detected. These findings suggest that the diversity of CD4 and CD8 TCRBV repertoires is decreased in patients with del22q11.2 syndrome, possibly as a result of either impaired thymic function and/or increased T-cell activation.

Clonotype tracking of TCR repertoires during chronic virus infections.

Human viral infections such as HIV and EBV typically evoke a strong and diverse CD8(+) T cell response. Relatively little is known about the extent to which TCR repertoire evolution occurs during viral infection or how repertoire evolution affects the efficacy of the CD8(+) T cell response. In this study we describe a general approach for tracking TCR repertoire evolution during viral infection. IFNgamma surface capture and MHC class I tetramer staining were independently used to isolate EBV-specific CD8(+) T cells from peripheral blood. Anchored RT-PCR and clonotype TCR repertoire analysis were performed immediately after isolating the cells. We find that the TCR repertoires of the IFNgamma-secreting and MHC class I tetramer staining populations were similar. In one subject a detailed analysis of the TCR repertoire during the first year of EBV infection was performed and over 600 TCR sequences targeting an EBV-immunodominant epitope were analyzed. Although some repertoire evolution occurred during the year, in general, the degree of repertoire drift was small. TCR repertoire analysis for an HIV-immunodominant epitope revealed a highly conserved amino acid motif in the Dbeta region of TCR that recognizes the epitope and suggested that T cell precursor frequency influences which epitopes are targeted early in HIV infection. This methodology, which allows one to sort antigen-specific T cells based on different functional assays and to obtain a snapshot of their TCR repertoire with relative ease, should lead to a richer understanding of the rules underlying antigen recognition and T cell evolution during viral infection.

Monitoring HIV-specific CD8+ T cell responses by intracellular cytokine production

Human immunodeficiency virus (HIV)-specific CD8+ T cells play an important role in controlling HIV infection. Accurate monitoring of these cells is crucial in determining the effects of HIV therapy and vaccine efficacy. Using an intracellular cytokine staining based assay, we are able to directly quantify functional HIV-specific CD8+ T cells. This assay is highly reproducible, and can be performed using both fresh and cryopreserved peripheral blood cells. Importantly, this assay can be used to examine multiple HIV-peptide epitopes simultaneously, and is independent of patient HLA haplotype. Here, we examine the HIV-specific CD8+ T cell response to 95 optimized HIV-derived cytotoxic T lymphocyte (CTL) epitopes in 21 HIV-infected patients of varying HLA haplotype, using peptide mixes and matrices. We find that when using mixes of multiple HIV peptides, the CD8+ T cell response to the mixture is equivalent to the sum of the responses to the individual peptides contained therein. Detailed comparison of the responses in patients suggests that most patients generate a diverse CD8+ T cell response, recognizing multiple HIV epitopes derived from HIV Gag, Pol, Env, or Nef. Although some patients sharing HLA alleles occasionally recognize common peptides, rarely are responses to those peptides dominant within the same group of patients. These results confirm our previous findings that the responses to single HIV-peptides are rarely representative of the entire HIV response.