In vitro priming of human lymphocytes. I. IL-2 and IL-4 requirements

Brams, Peter; Royston, Ivor; Boernert, Paula

doi:10.3233/hab-1993-4202

Cited by 9 publications

(5 citation statements)

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“…Although IL2 and IL4 had no influence per se on the heightened HCV RNA detection (Fig. 2), they were included in the Combo 5 cocktail since their presence is known to support T-and B-cell survival (Brams et al, 1993;Marrack et al, 1998). It was also evident that virus replication and expression were augmented to a variable degree in PBMCs from different individuals (see Figs 2 and 3) and that the magnitude of the response was usually lower in cases where the cells were HCV RNA reactive prior to the treatment.…”

Section: Discussionmentioning

confidence: 99%

Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells

Pham

MacParland

Coffin

et al. 2005

Journal of General Virology

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Considering growing evidence indicating that hepatitis C virus (HCV) replicates in lymphoid cells, establishment of a reliable and sensitive method for detection of HCV in these cells may provide means for monitoring the infection and the efficacy of sterilizing antiviral therapy. In this study, conditions for ex vivo augmentation and detection of the HCV genome in peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C (CHC) or after a sustained virological response (SVR) to antiviral treatment were assessed. Following stimulation with combinations of mitogens and/or cytokines, PBMCs and, in certain cases, affinity-purified T and B cells were examined for HCV positive-and negative-strand RNA by using RT-PCR followed by nucleic acid hybridization, while the presence of viral NS3 protein was determined by flow cytometry. HCV RNA augmentation was assessed by quantification of Southern and dot-blot hybridization signals. The results showed that treatment of peripheral lymphoid cells with mitogens stimulating T-and B-cell proliferation and with cytokines supporting their growth significantly increased HCV RNA detection in patients with both CHC and SVR. This enhancement was up to 100-fold for the HCV genome and fivefold for the NS3 protein compared with untreated cells. In conclusion, HCV RNA can be readily detected in circulating lymphoid cells in progressing hepatitis C and following SVR after ex vivo cell stimulation. As such, this method offers a new investigative tool to study HCV lymphotropism and to monitor virus presence during the course of HCV infection.

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Section: Discussionmentioning

confidence: 99%

Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells

Pham

MacParland

Coffin

et al. 2005

Journal of General Virology

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“…Responses by male spleen cells to intact LNCaP cells and PMA are possibly a consequence of an MLC type stimulation induced by HLA molecules or/and specific tumor antigens on the tumor cells and in the membrane preparation. Anti-PSA responses could only be measured from female cells supported with IL-2, strongly suggesting that IL-2 also is a critical factor for induction of significant non-MLC type responses, as previously shown (15). Formation of a mixed splenocyte culture was a help but not a prerequisite for induction of antigen specific responses.…”

Section: Discussionmentioning

confidence: 79%

“…This approach involved incubation of splenocytes with antigen to determine priming, with a parallel incubation of non-immunized splenocytes to evaluate nonspecific reactivity. Significant antigen-directed IgM responses were observed in supernatant taken day 10 and day 15 Factors affecting production of antibody to PMA and PSA Antigen-induced in vitro IgM response to a membrane preparation of prostate tumor (PMA) from male and female splenocytes are shown in figures 2 and 3, respectively. No IgG response were detected.…”

Section: Factors Affecting the Production Of Antibody To Intact Tumormentioning

confidence: 98%

“…Cells were distributed into the wells of a 24-well tissue culture plate at 3 x 10" cells/ml, 2 ml/well in RPMI-1640 supplemented with 10% FCS, 1% non-essential amino acid, 2 mM glutamine and 1 mM sodium pyruvate (RF-10+) (3) with or without 25 U/ml IL-2 (15). The immunizing antigen, PSA or PMA, was immediately added to the cultures at concentrations of 0, 0.5, 1.0 and 2.0 ¿tg/ml.…”

Section: In Vitro Immunizationmentioning

confidence: 99%

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Factors Affecting Production of Antibodies to Prostate Antigens by In Vitro-Primed Human Splenocytes

Bustria²,

Royston³

et al. 1993

Hybridoma

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Efficiency of immunization of splenocytes in vitro by three kinds of prostate antigen has been studied. Intact cells of the human prostate cell line LNCaP, a membrane preparation (PMA) of LNCaP cells, and prostate specific antigen (PSA) were used as antigens. Three different schemes of in vitro immunization were tested: male spleen cells vs female; single donor spleen cells vs. mixed lymphocyte culture (MLC); addition of exogenous IL-2 vs. no lymphokine addition. The supernatant antibodies were tested for reactivity to the immunizing antigen by ELISA. Subsequently hybridomas were generated by fusing the primed lymphocytes to a heteromyeloma cell line, K6H6/B5. Only antigen specific IgM responses could be detected. Intact LNCaP cells induced the highest responses from mixed lymphocyte cultures. PMA also induced the highest responses from mixed lymphocyte cultures of male origin, whereas both single donor and mixed donor spleen cell cultures of female origin responded to PMA. However, anti-PMA responses by mixed lymphocyte cultures of female cells were significantly augmented by addition of IL-2. PSA only induced specific responses from mixed female splenocyte cultures supported with IL-2. Several anti-PMA and -PSA antibodies were generated after somatic fusion of the in vitro primed cells. Two monoclonal IgG antibodies from LNCaP primed spleen cells could be competitively inhibited with tumor membrane antigen.

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“…Several research groups have developed various in vitro immunization procedures for the preparation of specific immortalized human B-lymphocytes using various fusion partners [ 14 , 15 ] or Epstein-Barr virus [ 16 – 18 ]. However, these techniques have a poor yield and reproducibility [ 19 – 21 ]. Furthermore, the protocols reported are complex since they require several steps for depletion or enrichment of various sub-populations [ 15 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Production of antigen-specific human IgGs by in vitro immunization

et al. 2016

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BackgroundWe previously developed in vitro immunization based on a fusion protein containing the transcriptional transactivator (Tat) of human immunodeficiency virus and a double domain, called ZZ, derived from protein A of Staphylococcus aureus. In this approach, naïve human peripheral blood mononuclear cells (PBMCs) trigger a specific IgM antibody (Ab) response in the presence of ZZTat. In the present study, we attempted to raise a specific IgG Ab response.ResultsWe found that PBMCs incubated with ZZTat and a mixture containing anti-CD40, IL4 and IL21 secrete anti-Tat IgG Abs in their supernatants, indicating that the cytokine cocktail provides an isotypic switch. Then, we deciphered the Tat determinant involved in the phenomenon and found that it is located in the region 22–57 and that, within this region, the cysteine-rich domain and the basic residues play a crucial role. Finally, we prepared a fusion protein containing a fragment derived from the NY-ESO-1 cancer/testis antigen (Ag) and showed that PBMCs incubated with ZZfNY-ESO-1Tat trigger a specific anti-fNY-ESO-1 IgG Ab response, which demonstrates the possibility of transferring immunizing ability to an Ag unrelated to Tat.ConclusionOur ZZTat-based in vitro immunization approach that offers the possibility to raise an IgG Ab response against NY-ESO-1 might represent a valuable first stage for the generation of fully human IgG specific Abs.

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In vitro priming of human lymphocytes. I. IL-2 and IL-4 requirements

Cited by 9 publications

References 0 publications

Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells

Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells

Factors Affecting Production of Antibodies to Prostate Antigens by In Vitro-Primed Human Splenocytes

Production of antigen-specific human IgGs by in vitro immunization

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