Dear Flowers, I am posting the responses to my question about Quantitation/Monitoring of CMV by Flow. Also I would like to mention, that for more detailed information and for possible additional questions interested parties should contact Lori Krueger from Beckman Technical support at (866)- 485-1270 # 2, # 1. I met Lori at the last CCS meeting and was able to get answers to all my questions. Regards. Irina Irina Grigorieva, PhD Director, Flow Cytometry Laboratory Northside Hospital, Atlanta, GA (404)- 851-6541 e-mail: irina.grigorieva@northside.com RESPONSES: Dear Irina, Sorry for the delay of my reply and thank you for your interest regarding this approach. The iTAg CMV kit is design to follow the CMV specific immune response after stem cell transplantation, in using the tetramer technology . Depending of HLA typing of the patient , the clinicians can make the follow up of the specific T cell recovery with the corresponding tetramer among a panel of 5 tetramers and alleles (iTAg CMV HLA A*0101; iTAg CMV HLA A*0201; iTAg CMV HLA B*0702; iTAg CMV HLA B*3501; and iTAg CMV HLA B*0801). The detection of the specific T cells after stem cell transplantation is done every two weeks until 1 year. This approach did not replace the CMV DNA detection by PCR, but allow to determine the need to give antiviral drugs. The following poster illustrate very well how use this method and show an example of patients. (See attached file: Poster CMV detection final version.pdf) Following results from a recent multicenter clinical trial, that allogeneic SCT patients who did not achieve a critical threshold of immune reconstitution of 7 cells /µL of CMV-specific CD8+ T cells, as measured by tetramers, within the first 65 days post transplant were 3.3 to 5.0 times more likely to develop recurrent or persistent CMV reactivation or other CMV-related complications. Patient's that showed rapid recovery (= 7 cells/ìL) of CMV-specific T cells were at lower risk of these complications. These data were not been already published at my knowledge. To have more details it could be better to discuss with Mark Thornbern about the sale strategy around this product. I hopping that this can help you. Do not hesitate to call me if you need more details. Christophe LE BOULAIRE Scientific and Technical Support Cellular Solution Beckman Coulter Marseille France Phone ++ 33 (0) 4 91 17 35 18 Fax ++ 33 (0) 4 91 17 27 53 cleboulaire@beckman.com www.Immunomics.com <<Poster CMV detection final version.pdf>> Dear Irina, I wasn't quite sure if you wanted to check new infections in immunosuppressed CMV- subjects given CMV+ donations. We have recently looked at primary HIV-1 infection, but found one patient with concurrent primary CMV infection (Zaunders et al, Blood, 2005, Sep 1;106(5):1660). These patients were investigated at, or soon after, the symptomatic phase. We found that such patients had a distinct subpopulation of CD4 that were CCR5+ and CD38 bright (very bright). In particular the primary CMV subject had something like 50% of his CD4 with this phenotype. Also, this patient had 5% of his CD4 made IFN-gamma by intracellular cytokine assay using whole CMV lysate as antigen, confirming that at least some of the large population of activated cells were CMV-specific ( I think they all are, but the assay doesn't pick them up) The problem is that you need a good machine (we use the LSR II) with very good sensitivity, and very fresh blood (< 2hr old) to see these cells. Possibly CCR5-PE on a good Calibur will be sensitive enough. We think these cells apoptose very rapidly after the blood is taken, or else CCR5 is rapidly down-regulated, making them hard to find normally. The high CD38 is very stable though. I saw someone suggested CD38, so I thought this may be interesting to you. Good luck, John Zaunders -- John Zaunders Senior Scientist, Centre for Immunology, St Vincent's Hospital, Darlinghurst, NSW, Australia Dear Dr. Grigorieva Use ProImmune's MHC Pentamers to detect antigen-specific T cells and as a new customer benefit from our limited time special offer of 15% off the 150-test quantity. ProImmune's Pro5 MHC Pentamer technology <http://www.proimmune.com/Direct/direct.php?e=1005P9&page=p_cat.htm&p=30342&CN=A4111262151*$_[I+IRI> leads the field in the sensitive detection and characterization of antigen-specific T cells using flow cytometry applications. MHC class I complexes are available specific for a wide range of infectious diseases such as Influenza, CMV, Hepatitis B & C, HIV, EBV as well as cancer-related epitopes. Custom Pentamers are also available for your individual research needs. Whether you are about to start epitope discovery and validation experiments or are already gathering data using other techniques or alternative multimer products, contact ProImmune to find out more about how we can help your research. Amanda Turner Marketing Operations Manager ProImmune Inc. Fairfax County BioAccelerator, Springfield, VA 22150, USA; Toll Free: +1 888 505 7765 www.proimmune.com <http://www.proimmune.com> Dear Irina, Dear Flowers, Although MHC Tetramer-based measurement of specific immune response is certainly of interest, I would like to point out that measuring the expression level of CD38 on CD8 bright (T) lymphocytes may also be of significant interest to monitor CMV infection in BM transplant patients. Although CD38 expression level quantitation is most popular in the context of HIV infection (increased in infectious periods and back to normal after effective therapy, see for ex. ref. 1), it is also very informative in other viral infections, as illustrated for infectious mononucleosis caused by EBV and/or CMV (Zidovec Lepej et al. , Ref 2). In this study, the high CD38 expression levels (Median 25,420 sABC - Min 3,252 - Max 37,262) measured were very different from normal values in healthy controls (Median 556 sABC - Min 350 - Max 988). Such a broadly reactive parameter would not be able to replace a specific assay such as iTAg TM MHC Tetramer CMV, but it may certainly be useful trying it to find out the appearance of a viral infection, monitor the evolution and check the efficiency of therapeutic regimens. Luckily enough, the appropriate tool (CellQuant CD38/CD8PE) is also available from the same vendor. If ever you have questions on that topic, I would be more than happy to share with you additionnal data we got from users in other viral infections as well as other useful parameters (Fas CD95 quantitation). Ref 1 : Benito J-M. et al., Differential upregulation of CD38 on different T-cell subsets may influence the ability to reconstitute CD4+ T cells under sucessful higly active antiretroviral therapy" J Acquir Immune Defic Syndrom 2005, 38 : 373-381. Ref 2 : Zidovec Lepej et al. Increased numbers of CD38 molecules on bright CD8+ T lymphocytes in infectious mononucleosis caused by Epstein-Barr virus infection. Clin Exp Immunol 2003; 133:384-390. Hope that helps Philippe Poncelet, PhD Director, Research and Technology BioCytex 140, Chemin de l'Armée d'Afrique 13010 Marseille France Tel: +33 (0) 4 96 12 20 40 Fax: +33 (0) 4 91 47 24 71 www.biocytex.fr This is NOT a replacement for CMV-PCR assays. We routinely monitor our BMT recipients by PCR to detect viral replication. We also monitor their immune reconstitution by CMV-specific pentamer assay (same principle as the Beckman Coulter kit). Frequently patients who have not yet reconstituted a T cell response to CMV remain CMV PCR negative but no-one has determined a level of CMV-reactive T cells which definitely protect from CMV reactivation, although some data are beginning to enter the literature. We have an ongoing trial where CMV-specific T cells are isolated from BMT donors and infused into recipients post BMT if they become PCR+ve and we know that very small numbers of specific T cells are required for protective immunity. These data will be presented at ASH in December. Call me if you want any more info. Regards Dr Mark W Lowdell PhD MRCPath Senior Lecturer / Director of Laboratory of Cellular Therapeutics, RFUCMS Tel - +44 (0) 20 7830 2183 Fax - +44 (0) 20 7830 2092 Dear Irina: I am not a flower but a hematologist, and I participated in the Beckman tetramer study at City of Hope. At present, immunologic monitoring alone is not considered sufficient to identify patients at risk for developing CMV disease (pneumonia, etc.). In clinic, the current standard practice should remain to be virologic monitoring such as CMV-PCR or pp65 antigenemia in deciding who and when to start preemptive anti-CMV therapy. This immunologic modality was developed to guide clinicians to better identify high (or low) risk patients for management in addition to the currently available tests and clinical risk factors. It is possible that in the future, we may be able to say that patients with high CMV tetramer values are at minimal or no risk for CMV disease and require no virologic monitoring. But at this time, the optimal use of the Beckman CMV tetramer needs to be further studied. I did not know the presentation was cancelled. But I would expect the updated data to be presented at various meetings in the near future. Please let me know if you have any questions. Best, Ryo Ryotaro Nakamura, MD City of Hope National Medical Center 1500 E. Duarte Road Duarte, CA 91010 Tel 626-359-8111, ext. 65285 Irina, We have been using home-prepared CMV tetramers for over 5 years in our laboratory on HSCT samples. My response would be that since the PCR and the tetramers are measuring two totally different things, any substitution is dangerous. One is measuring viral load in plasma and the other is measuring levels of CMV-specific T-cells. The two are related, but the relationship is indirect and complex. In my experience, an increase in CMV viremia is often followed by an expansion of CMV-specific CD8+ T cells, which often leads to a resolution of the viremia. However, since the viremia causes our HSCT physicans to prescribe prophylactic GCV treatment, it is hard to separate the effect of this GCV prophylaxis from any controlling effect of the CMV-specific CTL responses. We and others have published a few papers on this subject. Simon F. Lacey Ph.D. Assistant Research Scientist Laboratory of Vaccine Research Beckman Research Institute of the City of Hope Fox South Bld. City of Hope National Medical Center Duarte CA 91010-3000 Hi Irina, We are currently developing the CMV tetramer assay using the Beckman coulter Kit. We plan to offer this as a complete test packet for assessing immune competence of transplant patients. I am sorry I cannot give you any data at present since the test is still in development, but it sure can be used a robust method to quantify CMV specific Cytotoxic T cells in transplant patients and I will sure share some data with you when we get the test packet out for routine clinical use. Thanks Vijay Nandakumar, Cellular and Molecular Immunology Laboratory Mayo Clinic 200, 1st street SW, Rochester, MN, 55905 Ph: 507-284-2128 (Direct) Lab Secretary: 507-284-4055 Pager: 127-10415 Cell: 865-406-6788 Irina, This kit will only give you an indication of CMV responses to a single CMV peptide which is HLA-A2 restricted. If you read the manuscripts from Louis Picker (JEM 2005) and Florian Kern (JEM 2005), you will see firstly that measurement of a single response is not predictive of all responders or their total response to CMV; secondly, you will see that recognition of the HLA-A2 restricted peptide from this kit, which is from the CMV pp65 antigen, is actually predictive of BM transplant recipients who do poorly after failure, whereas recognition of the IE-1 protien is predictive of a better prognosis. Just because someone is HLA-A2 positive does not mean that they will respond to the HLA-A2 restricted CMV peptide from this kit. You would be far better off using intracellular cytokine staining, and either using whole inactivated CMV virus (which would be better for detecting CD4 responses), or a mixture of defined CD8 epitopes from CMV (or alternatively overlapping peptides covering pp65, IE1, and other ORFs, as described in the Picker manuscript). Just my 2 cents. Mike Michael R. Betts, Ph.D. Assistant Professor Department of Microbiology 522E Johnson Pavilion 3610 Hamilton Walk Philadelphia, PA 19104 (215) 573-2773 (office) (215) 746-6526 (lab) (215) 573-4446 (fax) This attachment - 'Poster CMV detection final version.pdf' - 891.49 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/93d75ab9b27692e634683b4462185e814d3247c2.pdfReceived on Tue Nov 1 17:57:40 2005
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