Evaluation of stabilized blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting

BACKGROUND: Exceptionally robust cell preparations are needed for quality assessment programs (QAPs) such as the International Program for Quality Assessment and Standardization for Immunological Measures (QASI) relevant to HIV/AIDS. A suitable product must withstand environmental stress related to transportation for a minimum of 6 days. The two objectives of this study are (1) to evaluate the performance of various commercial preparations with multicenter participation and (2) to evaluate the robustness of stabilized blood cell products. METHODS: Phase 1: The performance of stabilized blood cell products was evaluated in a multicenter QAP utilizing various staining procedures and flow cytometers. Absolute cell enumeration was achieved using single-platform T-cell subset methodology. Phase 2: The robustness of stabilized blood cell products was evaluated by monitoring T-cell subset values from samples stored at 4 degrees C, 22 degrees C, and 37 degrees C for up to 10 days. RESULTS: The largest interlaboratory variation in both absolute and relative T-cell values was 16% in samples with CD4 levels > or =400 cells per microliter and 21% in samples with CD4 levels <400 cells per microliter. Six preparations retained their phenotypic expression for 7 days at 4 degrees C and 22 degrees C. However, only two preparations remained stable for 4 days at 37 degrees C. CONCLUSION: Some stabilized cell preparations are more robust and therefore more suitable for quality assessment purposes.     

In vivo mutant frequency of thioguanine-resistant T-cells in the peripheral blood and lymph nodes of melanoma patients

T-cell activation by malignant melanoma would be anticipated to stimulate T-cell proliferation, which in turn has been associated with increasing the likelihood of somatic gene mutation. The purpose of this study was to test the hypothesis that in vivo hypoxanthine guanine phosphoribosyltransferase (hprt) mutant frequencies (MFs) are increased in peripheral blood T-cells from melanoma patients compared to normal controls. Assays were made of 48 peripheral blood samples from melanoma patients with stage 3 (13 patients) and stage 4 (35 patients) disease, 38 normal controls, and of nine tumor bearing lymph nodes. The mean hprt log(10)(MF) in patient peripheral blood was -4.77 (geometric mean hprt MF=17.0x10(-6)) compared to a mean hprt log(10)(MF) of -4.87 (geometric mean hprt MF=13.5x10(-6)) in controls. Although modest, this difference is statistically significant both by t-test (P=0.049) and after adjustment for covariates of age, gender, and cigarette smoking by regression analysis (P=0.001). Among the melanoma patients, the mean log(10)(MF) for the 17 patients who had received potentially genotoxic therapies was not significantly different from the mean log(10)(MF) for the 31 patients not receiving such therapies. The hprt MFs in the nine tumor bearing nodes were compared with MFs in peripheral blood from the same patients and revealed a non-significant (P=0.07) trend for increasing MFs in blood. Furthermore, analyses of T-cell receptor gene rearrangement patterns revealed hprt mutants originating from the same in vivo clone in both peripheral blood and a tumor-bearing node. The finding of elevated hprt MFs not entirely explained by genotoxic therapies in patients compared to controls can be explained either by hypermutability or in vivo T-cell activation. The similar MFs in peripheral blood and tumor bearing lymph nodes, as well as the finding of mutant representatives of the same in vivo T-cell clone in both locations, support monitoring peripheral blood to detect events in the nodes. If in vivo proliferation accounts for the current findings, the hprt deficient (hprt-) mutant fraction in blood may be enriched for T-cells that mediate the host immune response against malignant melanoma. Further studies will characterize the functional reactivity of hprt mutant isolates against melanoma-related antigens.     

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIV_mac251 infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4⁺ T-cell responses (mean, 5.9% ± 1.3% of all CD4⁺ T cells) and to a lesser extent SIV-specific CD8⁺ T-cell responses (mean, 0.7% ± 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4⁺ T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4⁺ T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted.     

Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.     

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25?% of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50?% of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

The specific targeting of immune regulation: T-cell responses against Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.     

Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.     

Dendritic cells: sentinels against pathogens

Dendritic cells (DCs) are the most potent antigen-presenting cells, and are regarded as "natural adjuvants" for the induction of primary T or T-dependent immunity. DCs in the peripheral sites capture and process antigens. Encounter of exogenous or endogenous stimuli mature the function of DCs, and they thus acquire T-cell stimulatory capacity and distinct chemotactic behavior which enables them to migrate to lymphoid tissue. In the secondary lymphoid organs, they present antigens to T- and B-cells and stimulate their proliferation. Dendritic cells are also involved in tolerance induction, in particular, to self antigens. DCs also play a key role in the transmission of many pathogens, and therefore may become targets for designing new therapies. DCs have been manipulated in vitro and in vivo for cancer immunotherapy. In this article, we provide a concise overview of DC biology and its current and future role in clinical settings.     

Intrinsic capacity of monocytes to produce cytokines ex vivo in patients with acute lymphoblastic leukaemia

Monocytic cytokine profiles of fifteen children with acute lymphoblastic leukaemia (ALL) were included to determine whether malignancy per se contributes to impaired cytokine profiles in vivo and ex vivo. The ex vivo tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) production was positively correlated with the monocyte number and with the number of intracellular TNF-alpha or IL-1beta positive cells in lipopolysaccharide (LPS)-stimulated MNC cultures. The mean ex vivo TNF-alpha and IL-1beta production per 1x10(4)monocytes in these cultures was not significantly different in children at diagnosis of ALL, at remission or in controls. High IL-10 plasma levels at diagnosis of ALL had no effect on the ex vivo TNF-alpha and IL-1beta production of monocytes in LPS stimulated MNC cultures. These results show that monocytes of ALL patients have a normal intrinsic capacity to produce cytokines ex vivo. However, the decreased monocyte number is responsible for the lower TNF-alpha and IL-1beta concentrations ex vivo upon LPS stimulation.     

Peripheral blood T-lymphocytes of patients with chronic B-cell lympholeukemia express Ia-like antigens

15.7 +/- 3.5% of sheep rosette-forming cells (SRFC) were isolated from the peripheral blood of 11 patients with B-cell chronic lympholeukemia (B-CLL). SRFC did not express surface immunoglobulins, antigens of nondifferentiated blasts and antigens of early T-cell precursors, while NK-cell antigen expression was low. 6 of 11 patients revealed 44.6 +/- 19.6% of Ia-like antigens in SRFC. Ia-like antigen expression was 4.8 +/- 0.6% in SRFC isolated from the peripheral blood of 19 healthy donors. The expression of Ia-like antigens in T cells of patients with B-CLL is suggested to be related to the activation of regulatory T-lymphocyte subpopulation.     

In vivo immunization against autologous glioblastoma-associated antigens

Summary A glioblastoma patient was immunized in vivo with a mixture of autologous and homologous glioblastoma cells coupled to adjuvant peptide and cord-factor analog. Immune activity of peripheral blood lymphocytes was measured in a short-term ⁵¹chromium-release assay against autologous tumor target cells. The patient developed direct cell-mediated cytotoxicity against tumor-associated antigens, which appeared to be T-cell mediated.     

Dendritic cells crosspresent antigens from live B16 cells more efficiently than from apoptotic cells and protect from melanoma in a therapeutic model

Dendritic cells (DC) are able to elicit anti-tumoral CD8(+) T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8(+) T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8(+) T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.     

Hepatitis C virus (HCV)-specific CD8+ cells produce transforming growth factor beta that can suppress HCV-specific T-cell responses

Hepatitis C virus (HCV)-specific T-cell responses are rarely detected in peripheral blood, especially in the presence of human immunodeficiency virus (HIV) coinfection. Based on recent evidence that T-regulatory cells may be increased in chronic HCV, we hypothesized that functional blockade of regulatory cells could raise HCV-specific responses and might be differentially regulated in the setting of HIV coinfection. Three groups of subjects were studied: HCV monoinfected, HCV-HIV coinfected, and healthy controls. Frequencies of peripheral T cells specific for peptides derived from HCV core, HIV type 1 p24, and recall antigens were analyzed by gamma interferon (IFN-gamma) enzyme-linked immuno-spot assay. HCV-specific T-cell responses were very weak in groups with HCV and HCV-HIV infections. Addition of blocking antibodies against transforming growth factor beta1 (TGF-beta1), -2, and -3 and interleukin-10 specifically increased the HCV-specific T-cell responses in both infected groups; however, this increase was attenuated in the group with HCV-HIV coinfection compared to HCV infection alone. No increase in recall antigen- or HIV-specific responses was observed. Flow cytometric sorter analysis demonstrated that regulatory-associated cytokines were produced by HCV-specific CD3(+)CD8(+)CD25(-) cells. Enhancement of the IFN-gamma effect was observed for both CD4 and CD8 T cells and was mediated primarily by TGF-beta1, -2, and -3 neutralization. In conclusion, blockade of TGF-beta secretion could enhance peripheral HCV-specific T-cell responses even in the presence of HIV coinfection.     

Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.     

Analysis of T-cell receptor repertoire in peripheral blood of patients with pancreatic cancer and other pancreatic diseases

Pancreatic cancer (PC) has been the fourth cancer-related death worldwide, diagnosed at an unresectable stage due to its rapid progression and few symptoms of this disease at early stages. The aim of this study was to determine the association between the diversity of T-cell receptor (TCR) repertoire and clinicopathological characteristics of patients with PC and other benign pancreatic diseases. In order to make a comprehensive analysis the TCR repertoire, high-throughput sequencing was used to differentiate complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 3 PC, 3 chronic pancreatitis, 3 pancreatic cystic lesions and 3 pancreatic neuroendocrine tumour patients. We found that there were significant differences related to TCR repertoire between PC and other pancreatic diseases, and PC is a relatively immunosuppressive tumour. Changes of peripheral TCR repertoire may be used to predict the progression of PC and the response to immunotherapy. And there may exist novel-specific antigens in PC patients which could be used to design targeting immunotherapy in the nearly future.     

Circumventing T-cell tolerance to tumour antigens.

During past decades, many attempts have been made to induce or enhance tumour-specific T-cell immunity in cancer patients by vaccination. However, it has become apparent that in a large number of cases the naturally occurring tumour-specific T-cell repertoire is of low affinity and therefore inefficient in mediating tumour rejection. Because of the potential therapeutic value of high affinity TCRs with tumour/lineage specificities, we set out to develop a number of new technologies that can be used to create improved tumour-specific T-cell immunity. These strategies entail: (i) the efficient expansion of low affinity T cells specific for self antigens through the use of variant peptides with improved TCR-binding characteristics; (ii) a retroviral library-based technology to improve the affinity of (self-specific) T-cell receptors in vitro, and (iii) proof of principle for the feasibility of TCR gene transfer as a means to generate T-cell populations with a desired antigen-specificity in vivo. Collectively this toolbox should allow us to create improved T-cell receptors for human tumour antigens, which can subsequently be used to impose tumour-reactivity on to peripheral T cells.     

Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells

A major focus of cancer immunotherapy is to develop strategies to induce T-cell responses through presentation of tumor antigens by dendritic cells (DCs). Current vaccines are limited in their ability to efficiently transfer antigens to DCs in vivo. Ex vivo-generated DCs can be efficiently loaded with antigen but after reinjection, few DCs traffic to secondary lymphoid organs, the critical sites for antigen presentation. To enhance efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem-progenitor cells (HSCs) with a model tumor antigen and then transplanted the gene-modified cells into irradiated recipient mice, which resulted in efficient expression of the transgene in a large proportion of donor derived DCs in lymphoid organs. The combination of bone marrow transplantation (BMT) using transduced HSCs, systemic agents that generate and activate DCs, and mature T-cell infusion resulted in substantial expansion and activation of antigen-specific T cells. This tripartite strategy provided potent antigen-specific immunotherapy for an aggressive established tumor.     

Increased peripheral blood gamma delta T-cells in patients with lymphoid neoplasia: A diagnostic dilemma in flow cytometry

We have observed increased numbers of non-neoplastic gammadelta-T-cells in the peripheral blood of a series of patients with non-Hodgkin's lymphoma not of gammadelta-T-cell origin. The majority of normal gammadelta-T-cells are negative for surface CD4 and CD8 and a subpopulation does not express CD5, two immunophenotypic findings strongly suggestive of neoplasia in alpha beta T-cells. In addition, they express cytotoxic T-cell/Natural killer cell antigens. In this study, up to 22% of PBLs were CD4 and CD8 negative gammadelta-T-cells and up to 33% PBLs were CD5 negative gammadelta-T-cells. In addition, as high as 42% of PBLS were gammadelta-T-cells expressing cytotoxic T-cell/Natural killer cell antigens, suggestive of a large granular lymphoproliferative disorder. Failure to recognize that these are normal gammadelta-T-cells could lead to the erroneous diagnosis of peripheral blood involvement with a T-cell neoplasm, especially in the setting of a history of non-Hodgkin's lymphoma. Cytometry (Comm. Clin. Cytometry) 38:280-285, 1999. Published 1999 Wiley-Liss, Inc.