CD8 Compartment Research Articles

Distinct Kinetics of Gag-Specific CD4+ and CD8+ T Cell Responses during Acute HIV-1 Infection

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.

Expansion of a CD8+PD-1+ Replicative Senescence Phenotype in Early Stage CLL Patients Is Associated with Inverted CD4:CD8 Ratios and Disease Progression

Patients with chronic lymphocytic leukemia (CLL) display immune deficiency that is most obvious in advanced stage disease. Here we investigated whether this immune dysfunction plays a pathologic role in the progression of early stage disease patients. We carried out eight-color immunophenotyping analysis in a cohort of 110 untreated early stage CLL patients and 22 age-matched healthy donors and correlated our findings with clinical outcome data. We found a significant reduction in naive CD4(+) and CD8(+) T cells in CLL patients. Only the CD4(+) subset showed significantly increased effector memory cells (T(EM) and T(EMRA)) in the whole cohort (P = 0.004 and P = 0.04, respectively). However, patients with inverted CD4:CD8 ratios (52 of 110) showed preferential expansion of the CD8 compartment, with a skewing of CD8(+) T(EMRA) (P = 0.03) coupled with increased percentage of CD57(+)CD28(-)CD27(-) T cells (P = 0.008) and PD-1 positivity (P = 0.027), consistent with a replicative senescence phenotype. Furthermore, inverted CD4:CD8 ratios were associated with shorter lymphocyte doubling time (P = 0.03), shorter time to first treatment (P = 0.03), and reduced progression-free survival (P = 0.005). Our data show that the emergence of CD8(+)PD-1(+) replicative senescence phenotype in early stage CLL patients is associated with more aggressive clinical disease. Importantly, these findings were independent of tumor cell prognostic markers and could not be accounted for by patient age, changes in regulatory T-cell frequency, or cytomegalovirus serostatus (n = 217).

Induction of contact‐dependent CD8+ regulatory T cells through stimulation with staphylococcal and streptococcal superantigens*

The bacterial superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes are potent stimulators of polyclonal T-cell proliferation. They are the causes of toxic shock syndrome but also induce CD25(+) FOXP3(+) regulatory cells in the CD4 compartment. Several studies have recently described different forms of antigen-induced regulatory CD8(+) T cells in the context of inflammatory diseases and chronic viral infections. In this paper we show that bacterial superantigens are potent inducers of human regulatory CD8(+) T cells. We used four prototypic superantigens of S.aureus (toxic shock syndrome toxin-1 and staphylococcal enterotoxin A) and Str.pyogenes (streptococcal pyrogenic exotoxins A and K/L). At concentrations below 1 ng/ml each toxin triggers concentration-dependent T-cell receptor Vβ-specific expression of CD25 and FOXP3 on CD8(+) T cells. This effect is independent of CD4(+) T-cell help but requires antigen-presenting cells for maximum effect. The cells also express the activation/regulatory markers cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumour necrosis factor receptor-related protein and skin homing adhesins CD103 and cutaneous lymphocyte-associated antigen. Superantigen-induced CD25(+) FOXP3(+) CD8(+) T cells were as potent as freshly prepared naturally occurring CD4(+) regulatory T cells in suppressing proliferation of CD4(+) CD25(-) T cells in response to anti-CD3 stimulation. Although superantigen-induced CD8(+) CD25(+) FOXP3(+) express interleukin-10 and interferon-γ their suppressive function is cell contact dependent. Our findings indicate that regulatory CD8(+) T cells may be a feature of acute bacterial infections contributing to immune evasion by the microbe and disease pathogenesis. The presence and magnitude of regulatory CD8(+) T-cell responses may represent a novel biomarker in such infections. Superantigen-induced regulatory CD8(+) T cells also have therapeutic potential.

The blood of healthy individuals exhibits CD8 T cells with a highly altered TCR Vß repertoire but with an unmodified phenotype

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25-52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ+ T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8+ T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8+ T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

The Blood of Healthy Individuals Exhibits CD8 T Cells with a Highly Altered TCR Vb Repertoire but with an Unmodified Phenotype

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25–52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ+ T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8+ T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8+ T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

Ten-Color flow cytometry reveals distinct patterns of expression of CD124 and CD126 by developing thymocytes

BackgroundWe have developed a 12-parameter/10-color flow cytometric staining method for the simultaneous detection and characterization of 21 mouse thymocyte subpopulations that represent discreet stages of T cell development. To demonstrate the utility of this method, we assessed cytokine receptor expression on mouse thymocyte subsets. These experiments revealed distinct patterns of surface expression of receptors for the cytokines IL-4 and IL-6.ResultsThe IL-4 receptor α chain (CD124) was highly expressed on the earliest thymocyte subsets, then downregulated prior to T cell receptor β-selection and finally upregulated in the CD4/CD8 double positive cells prior to positive selection. The IL-6 receptor α chain (CD126) showed a different pattern of expression. It was expressed on the most mature subsets within the CD4 and CD8 single positive (SP) compartments and was absent on all other thymocytes with the exception of a very small cKit-CD4-CD8- population. Intracellular staining of SP thymocytes for phosphorylated STAT-1 demonstrated that IL-6 signaling was confined to the most mature SP subsets.ConclusionsThis 12-parameter staining methodology uses only commercially available fluorochrome-coupled monoclonal antibodies and therefore could be employed by any investigator with access to a 4-laser flow cytometer. This novel staining scheme allowed us to easily phenotype thymocyte subpopulations that span across development, from the early thymic progenitors (ETPs) to the most mature subsets of the CD4 and CD8 single positive populations.

CD4+ and CD8+ T cell- and IL-17-mediated protection against Entamoeba histolytica induced by a recombinant vaccine

Amebiasis in the murine model can be prevented by vaccination with the Gal/GalNAc lectin through a T cell-dependent mechanism. In this work we further decipher the mechanism of this protection. Mice vaccinated with the recombinant “LecA” fragment of the Gal/GalNAc lectin with alum were capable of transferring protection to naïve recipients by both CD4+ T cells and surprisingly CD8+ T cells. We then examined the cytokine profile of these cells. CD4+ T cells from PBMC of LecA-alum vaccinated mice were observed to be a major source of IFN-γ, known to be a protective cytokine with this vaccine. In contrast, CD8+ T cells produced relatively little IFN-γ but more IL-17 than the CD4 compartment. We thus examined the role of IL-17 in vaccine mediated protection and found through neutralization experiments that this cytokine contributed to LecA-alum vaccine protection. In addition we examined whether these cells exhibited direct amebicidal activity in vitro and found that both populations had amebicidal activity at high concentrations (1000:1) but CD8+ T cells appeared more potent, capable of cytotoxicity at a 100:1 ratio. In conclusion, both CD4 and CD8 T cells exert protection with this amebiasis vaccine. The mechanism of CD8 T cell-mediated protection may include direct amebicidal activity and/or IL-17 production. Both IL-17 and IFN-γ are useful surrogates for immune protection.

Autoproliferation Contributes to Clonal Expansion and Changes In Cellular Topography In T Cell Large Granular Lymphocyte (LGL) Leukemia

AbstractAbstract 1373 Introduction:LGL leukemia is associated with cytopenias and expansion of clonally-derived mature cytotoxic CD8+ lymphocytes. The etiology of LGL leukemia is currently unknown, however, T cell activation, loss of lymph node homing receptor L-selectin (CD62L), and increased accumulation of T cells in the bone marrow may lead to suppressed blood cell production. The broad resistance to Fas (CD95) apoptotic signals has lead to the hypothesis that amplification of clonal cells occurs through apoptosis resistance. However, the proliferative history has not been carefully studied. To define possible mechanism of LGL leukemia expansion, T cell phenotype, proliferative history, and functional-related surface marker expression were analyzed. Methods:Peripheral blood mononuclear cells (PBMCs) were obtained from 16 LGL leukemia patients that met diagnostic criteria based on the presence of clonal aβ T cells and >300 cells/ml CD3+/CD57+ T cells in the peripheral blood. Samples were obtained from 10 age-matched healthy individuals from the Southwest Florida Blood Services for comparisons. Multi-analyte flow cytometry was conducted for expression of CD3, CD4/8, CD45RA, CD62L, CD27, CD28, CD25, CD127, IL15Ra, IL21a, CCR7 (all antibodies from BD Biosciences). The proliferative index was determined by Ki67 expression in fixed and permeabilized cells (BD Biosciences) and the proliferative history in vivo was assessed by T-cell-receptor excision circle (TREC) measurement using real-time quantitative PCR (qRT-PCR) in sorted CD4+ and CD8+ T cells. TRECs are episomal fragments generated during TCR gene rearrangements that fail to transfer to daughter cells and thus diminish with each population doubling that reflects the in vivo proliferative history. Results:Compared to healthy controls, significantly fewer CD8+ naïve cells (CD45RA+/CD62L+, 8.4 ± 10.8 vs 24.48 ± 11.99, p=0.003) and higher CD8+ terminal effector memory (TEM) T cells (CD45RA+/CD62L-, 67.74 ± 28.75 vs 39.33 ± 11.32, p=0.007) were observed in the peripheral blood. In contrast, the percentage of CD4+ naïve and memory cells (naïve, central memory, effector memory, and terminal effector memory based on CD45RA and CD62L expression) was similar in patients as compared to controls. The expression of CD27 (31.32 ± 34.64 vs 71.73 ± 20.63, p=0.003) and CD28 (31.38 ± 31.91 vs 70.02 ± 22.93, p=0.002) were lower in CD8+ T cell from patients with LGL leukemia and this reduction predominated within the TEM population (17.63±24.5 vs 70.98±22.5 for CD27, p<0.0001 and 13±20.5 vs 69.43± 21.59 for CD28, p<0.0001). Loss of these markers is consistent with prior antigen activation. There was no difference in CD25 (IL2Ra, p=0.2) expression on CD4+ or CD8+ T cells, but CD127 (IL7Ra, p=0.001), IL15Ra, and IL21Ra (p=0.15) were overexpressed in TEM CD8+ T cell in patients vs controls. All of these cytokine receptors belong to the IL2Rβg-common cytokine receptor superfamily that mediates homeostatic proliferation. In CD8+ T cells in patients, the IL-21Ra was also overexpressed in naïve, central and effector memory T cells. The topography of the expanded CD8+ T cell population was therefore consistent with overexpression of activation markers and proliferation-associated cytokine receptors. Therefore, we next analyzed Ki67 expression and TREC DNA copy number to quantify actively dividing cells and determine the proliferative history, respectively. We found that LGL leukemia patients have more actively dividing CD8+ TEM T cells compared to controls (3.2 ± 3.12 in patients vs 0.44 ± 0.44 in controls, p=0.001). Moreover, the TREC copy number in CD8+ T cells was statistically higher in healthy individuals after adjusting for age (177.54 ± 232 in patients vs 1015 ± 951 in controls, p=0.019). These results show that CD8+ cells in the peripheral compartment have undergone more population doublings in vivo compared to healthy donors. In contrast, the TREC copies in CD4+ T-cells were similar between LGL patients and controls (534.4 ± 644 in patients vs 348.78 ± 248.16 in controls, p>0.05) demonstrating selective cellular proliferation within the CD8 compartment. Conclusions:CD8+ T- cells are undergoing robust cellular activation, contraction in repertoire diversity, and enhanced endogenous proliferation in patients with LGL leukemia. Collectively, these results suggest that clonal expansion is at least partially mediated through autoproliferation in T-LGL leukemia. Disclosures:No relevant conflicts of interest to declare.

Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution

Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αβ-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future.

Increased generation of CD8 single positive cells in SOCS1-deficient thymus does not proportionately increase their export

Mice lacking suppressor of cytokine signaling 1 (SOCS1) accumulate CD8+ T lymphocytes in the thymus and in the periphery. Whereas IL-7 and IL-15 promote the generation of CD8 single positive (SP) thymocytes, IL-15 drives the expansion of CD8 T cells in the periphery. Here, we investigated whether increased production of CD8 SP thymocytes is accompanied by their increased export in SOCS1-deficient mice. In vivo labeling with bromodeoxyuridine showed increased cycling of CD8 SP thymocytes in SOCS1-deficient mice. However, SOCS1-deficient thymi contained increased proportion of CD24loCD69lo SP thymocytes as well as increased expression of Qa-2 in both CD4 and CD8 SP compartments. Analysis of recent thymic emigrants (RTE) following intrathymic labeling with fluorescein isothiocyanate revealed less efficient export of CD8 RTEs from SOCS1-deficient thymi and comparable CD4:CD8 ratio among RTEs in SOCS1-null and control mice. These findings show that the rate of export of CD8 SP thymocytes is not proportional to their generation in SOCS1-deficient thymi and suggest the existence of homeostatic mechanisms controlling the egress of CD8 T cells.

CD4 T-cell hyperactivation and susceptibility to cell death determine poor CD4 T-cell recovery during suppressive HAART

The failure to increase CD4 T-cell counts in some HAART-treated HIV-infected patients with satisfactory virological responses has been related to low CD4 T-cell production, high turnover and death. However, the relative contribution of these factors is still unclear, strongly limiting the definition of appropriate therapeutic strategies for these patients. A cross-sectional study was designed to evaluate the contribution of thymic activity, microbial translocation, cellular activation and death to CD4 T-cell recovery. We included 230 HIV-infected individuals on suppressive HAART (>2 years); 95 of them were considered 'discordant' (CD4 T-cell count <350 cells/mul) and 135 were considered 'concordant'. Comparative and logistic regression analyses were performed. Discordant patients showed higher levels of activated [human leukocyte antigen (HLA)-DRCD95 and CD38CD45RA] cells in both the CD8 and CD4 T-cell compartments. Notably, the most significant differences were observed in CD4 T cells. Discordant patients showed lower naive CD4 T-cell production (CD45RACD31 cells), higher spontaneous ex-vivo CD4 T-cell death and higher plasma levels of soluble CD14. Multivariate analysis showed that activation and death of CD4 T cells, along with nadir CD4 T-cell counts, were the only predictive factors for poor immune recovery. Moreover, the low correlations found between CD4 T-cell activation or death with thymic output and bacterial translocation suggest that additional factors modulate cellular activation and death and, in turn, CD4 T-cell recovery. CD4 T-cell repopulation during HAART is determined by CD4 T-cell activation and death. Therefore, strategies aimed to reduce these parameters should be envisaged to treat discordant patients.

Bystander activation of CD4+ T cells

Bystander activation of T cells, i.e. the stimulation of unrelated (heterologous) T cells by cytokines during an Ag-specific T-cell response, has been best described for CD8(+) T cells. In the CD8(+) compartment, the release of IFN and IFN-inducers leads to the production of IL-15, which mediates the proliferation of CD8(+) T cells, notably memory-phenotype CD8(+) T cells. CD4(+) T cells also undergo bystander activation, however, the signals inducing this Ag-nonspecific stimulation of CD4(+) T cells are less well known. A study in this issue of the European Journal of Immunology sheds light on this aspect, suggesting that common gamma-chain cytokines including IL-2 might be involved in bystander activation of CD4(+) T cells.

Antigen dose differentially controls the induction of specific Treg and Teff populations (49.5)

Abstract The concept that Th1/Th2 differentiation may be affected by antigen dose was introduced a decade ago (O’Garra, Bottomly). Recently, we reported that antigen dose also controls Treg induction whereby low antigen doses elicit weak signals via the TCR/Akt/mTOR pathway, allowing Foxp3 expression and expansion of Treg. METHODS: We expanded our studies on antigen dose effects to include regulatory and effector populations that have been recently added to the CD4+ repertoire. Purified CD4+ T cells from BDC2.5 TCR transgenic mice were stimulated by DC pulsed with increasing doses of peptide antigen. After 7 days of priming, T cells were re-stimulated with anti-CD3 and anti-CD28 and analyzed for cytokine production and Foxp3 expression. RESULTS: We found that altering the antigen dose qualitatively affected the CD4+ T cell response in a hierarchical manner. Low dose stimulation led to Foxp3+ Treg expansion; intermediate doses induced IL-13+ Th2 and IL-10+ Tr1 cells; and higher antigen doses favored Th1/Th17 responses. In addition, when low-dose-stimulated T cells were subsequently re-stimulated with a high antigen dose, the Th1/Th17 response remained diminished. CONCLUSIONS: Our results indicate that Tr1, Th17 and Treg are included in the repertoire of CD4+ T cells that are affected by antigen dose. These findings have broad implications for vaccine design and we are in the process of determining the in vivo significance of these qualitative changes to the CD4 compartment.

Chronological and immunohistochemical characterization of the mammary immunoinflammatory response in experimental caprine contagious agalactia

To explore the pathogenesis of caprine contagious agalactia (CA), we assessed the ability of Mycoplasma agalactiae ( Ma) to modulate the immune system in host tissues by immunohistochemically and chronologically characterizing the main cell subsets present during the mammary immunoinflammatory response. Fifteen lactating goats were inoculated with 10 10 colony-forming units (cfu) of Ma and killed 5, 15 or 45 days post-inoculation (dpi). Blood was taken before necropsy to determine antibodies and milk to determine mycoplasma number. Cells in mammary tissue expressing lysozyme, myeloid-histiocyte antigen (Mac387), major histocompatibility complex class II antigen , immunoglobulin G (IgG), IgA, and CD3, CD4 and CD8 lymphocytes were determined by immunohistochemistry. Results indicate an innate immune response in animals sacrificed at 5 dpi, characterized by an abundance of Mac387+ and lysozyme+ cells, that was unable to block or control Ma infection. Elevated numbers of all the cell subsets of the specific immune response (MHC-II+, IgG+, IgA+, CD3+, CD4+ and CD8+ cells) were observed during the subacute stage of the inflammatory process, represented by the 15 dpi group. However, these findings could not be correlated with an intense antibody response in blood. The chronic stage of the inflammatory process observed in the goats killed at 45 dpi was mainly characterized by expansion of the CD8 compartment at the expense of the CD4 subset leading to a reduced CD4/CD8 ratio. These results contribute to establishing the basic morphological and immunohistochemical characterization of the local immune response against Ma in the goat's mammary gland.

Early expression of stem cell-associated genes within the CD8 compartment after treatment with a tumor vaccine

Using a mouse neuroblastoma cell line, we have demonstrated that vaccination of tumor-free mice with a cell-based vaccine leads to productive immunity and resistance to tumor challenge, while vaccination of tumor-bearing mice does not. The T cell immunity induced by this vaccine, as measured by in vitro assays, is amplified by the depletion of Treg. Our goal is to understand this barrier to the development of protective cellular immunity. mRNA microarray analyses of CD8 + T cells from naïve or tumor-bearing mice undergoing vaccination were carried out with or without administering anti-CD25 antibody. Gene-expression pathway analysis revealed the presence of CD8 + T cells expressing stem cell-associated genes early after induction of productive anti-tumor immunity in tumor-free mice, prior to any phenotypic changes, but not in tumor-bearing mice. These data demonstrate that early after the induction of productive immune response, cells within the CD8 + T cell compartment adopt a stem cell-related genetic phenotype that correlates with increased anti-tumor function.

Peptide-Specific, TCR-α–Driven, Coreceptor-Independent Negative Selection in TCR α-Chain Transgenic Mice

As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCR signaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.

Furin-Processed Antigens Targeted to the Secretory Route Elicit Functional TAP1−/−CD8+ T Lymphocytes In Vivo

Most pathogen-derived peptides recognized by CD8+ CTL are produced by proteasomes and delivered to the endoplasmic reticulum by the TAP transporters associated with Ag processing. Alternative proteases also produce antigenic peptides, but their actual relevance is unclear. There is a need to quantify the contribution of these supplementary pathways in vitro and in vivo. A well-defined TAP-independent secretory route of Ag processing involves the trans-Golgi network protease furin. Quantitation of this route by using OVA constructs encoded by vaccinia viruses indicates that it provides approximately one-third of all surface complexes of peptide and MHC class I molecules. Generation of the epitope carboxyl terminus is a dramatic rate-limiting step, since bypassing it increased efficiency by at least 1000-fold. Notably, the secretory construct activated a similar percentage of Ag-specific CD8+ T cells in wild type as in TAP1-deficient mice, which allow only secretory routes but which have a 10- to 20-fold smaller CD8 compartment. Moreover, these TAP1(-/-) OVA-specific CD8+ T lymphocytes accomplished elimination of epitope-bearing cells in vivo. The results obtained with this experimental system underscore the potential of secretory pathways of MHC class I Ag presentation to elicit functional CD8+ T lymphocytes in vivo and support the hypothesis that noncytosolic processing mechanisms may compensate in vivo for the lack of proteasome participation in Ag processing in persons genetically deficient in TAP and thus contribute to pathogen control.

HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation

HIV persists in a reservoir of latently infected CD4(+) T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in T(CM) cells in subjects showing reconstitution of the CD4(+) compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in T(TM) cells from aviremic individuals with low CD4(+) counts and higher amounts of interleukin-7-mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.

CLTA‐4 blockade in vivo promotes the generation of short‐lived effector CD8+ T cells and a more persistent central memory CD4+ T cell response

Previously, we examined the effects of in vivo CTLA-4 blockade using a fully human monoclonal antibody as a part of a DNA vaccination regimen in cynomolgus macaques (Macaca fascicularis). We observed that while the antibody had little effect on the IFN-gamma ELISpot response, CTLA-4 blockade enhanced antigen-specific cellular proliferation in both CD4(+) and CD8(+)T-cell compartments. We examine the specific effects of CTLA-4 blockade on memory T-cell compartments following the third immunization and 10 months following a fourth immunization, during the memory phase of the immune response. CLTA-4 blockade enhanced CD4(+) and CD8(+) central memory (CD28(hi), CD95(hi)) T-cell responses as well as a short-lived CD8(+) effector (CD28(lo), CD95(hi)) T-cell response. These data suggest differing effects of CTLA-4 blockade on CD4(+) and CD8(+) T cells with implications on the clinical use of anti-CTLA-4 antibodies for enhancement of vaccine strategies or treatment of human disease.

Minimal Residual Disease (MRD) and T/NK Cell Dynamics during Fludarabine, Cyclophosphamide Plus Rituximab (FCR) Followed by Fludarabine Plus Rituximab (FR) and Remission Maintenance Therapy with Rituximab in Previously Untreated B-Chronic Lymphocytic Leukemia (B-CLL): Riskfactor Stratification in the Chairos Study

Based on the efficacy of Fludarabine, Cyclophosphamide plus Rituximab (FCR) as first-line therapy for chronic lymphocytic leukemia (CLL), we conducted a non-randomized multi-center phase II trial with early brief intensive induction with FCR for 3 cycles followed by fludarabine/rituximab for 3 cycles. In addition patients received a 2 year 3-monthly rituximab maintenance regimen. The trial recruited 42 patients. Clinical baseline data, clinical response results and toxicity of a planned first interim analysis were reported previously (Egle et al, ASH 2007). We now present the data from flow cytometric minimal residual disease (MRD) monitoring during the study in combination with the performed risk factor analyses and report on the findings of detailed analysis of T-cell and NK cell dynamics during the induction phase of the trial. From the previously reported high number of CRs (94% after complete 6 cycle induction) 64% were MRD negative by flow cytometric definition (less than 10−3 G/L CLL cells). All non CR patients were correctly called as MRD positive. Less than 0.1 G/L CLL cells after 3 cycles of FCR identified all patients achieving MDR negativity after the complete induction regimen. CD30 risk, as well as age, FISH cytogenetic analysis and analysis of IgVH mutation status were analysed for their impact on MRD dynamics. We could not find any difference in MRD responses between CD38 high and low risk groups or between age groups (> and <65a). Neither did abberations found in FISH cytogenetics predict the MRD responses and all CLL patients harboring an 11q deletion achieved MRD negativity. Interestingly, we found the only predictive factor to be the IgVH mutation status with 15 unmutated CLL patients significantly less likely to achieve MRD negativity after induction treatment (p=0,025). Especially, three patients with rearranged IgVH 3–21 did poorly, suggesting that these patients may need a different therapeutic approach. Six months of Rituximab maintenance therapy were able to convert 37% of patients from MRD positive state after remission induction to a MRD negative state. T and NK cell numbers and ratios were followed during therapy. In addition T cell subsets and their CD38 expression status were analysed. While absolute T and NK/NKT cell numbers decreased dramatically during treatment (CD4 and CD8 cell numbers decreased to medians of less than 100/μl after 6 cycles of induction treatment), a count of less than 200 CD4 cells/μl after 3 cycles of FCR was found to be predictive of MRD negativity. In addition, we observed an earlier recovery of NK and NKT cells after the end of induction treatment leading to an inverted NK(T) to T cell ratio early during maintenance treatment. This may have implications for NK-dependent ADCC during rituximab maintenance therapy. Regarding the CD4 and CD8 compartment we observed in addition to the reduction in absolute numbers a marked shift of T cell subsets. Naïve T cells were more completely depleted by FCR/FR treatment than T cells from the memory compartment, with central memory (CM) CD4 cells increasing from a median of 49% to 79%. This was less pronounced in the CD8 compartment. CD38 expression on T cells was monitored, because our group found that this was predictive of CLL course (Tinhofer Blood 2006). Interestingly we found a marked increase of the proportional representation of CD38+ CD8 and CD4 T cells in the T cell compartment with a more pronounced effect observed in the CD8 compartment. CD8 T cell counts were also quicker to recover during rituximab maintenance therapy. While these data point to essential caveats regarding the immunosuppressive toxicity of the treatment, they may also point to a window of opportunity regarding the modification of T cell responses during the phase of immune reconstitution after FCR/FR therapy. In conclusion we find that only IgVH mutation status was predictive of achievement of MRD negativity and we could show significant skewing of T cell and NK(T) cell subsets during chemoimmunotherapy. A better understanding of these T/NK cell dynamics may lead to the development of improved targeting of the immune recovery phase after chemotherapy using immunomodulating maintenance strategies.