Jan-April 2008:
Chen, C. I., H. T. Maecker, and P. P. Lee. 2008. Development and dynamics of robust T cell responses to CML under imatinib treatment. Blood. Link to PubMed abstract.
This paper describes the unexpected finding of large CD4+ TNF responses to CML in patients treated with imatinib (Gleevec). The responses were often transient and included more cells making TNF than IFNg, suggesting a defect in antigen presentation or signalling that prevents a durable and truly successful response to the cancer cells.
Emu, B., E. Sinclair, H. Hatano, A. Ferre, B. Shacklett, J. N. Martin, J. M. McCune, and S. G. Deeks. 2008. HLA Class I-Restricted T Cell Responses May Contribute to the Control of HIV Infection, but Such Responses are Not Always Necessary for Long-term Virus Control. Journal of virology. Link to PubMed abstract.
Our colleagues at UCSF have correlated the presence of protective HLA alleles with CD8 T cell responses to Gag in a large group of "elite controllers". While about 2/3 of these individuals have such responses, the remainder have low or negative CD8 T cell responses to a Gag peptide pool, and the authors suggest that other mechanisms may protect these individuals from progression. See the evaluation in Faculty of 1000 Medicine for more details.
Jacobson, M. A., Q. X. Tan, V. Girling, C. Poon, M. Van Natta, D. A. Jabs, M. Inokuma, H. T. Maecker, B. Bredt, and E. Sinclair. 2008. Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS. Clin Infect Dis 46:458-466. Link to PubMed abstract.
A number of studies have now shown that CD4+ and/or CD8+ T cell responses correlate with recovery from CMV retinitis in HIV+ individuals. This study attempts to look longitudinally, using previously frozen specimens, at patients who did or did not develop CMV retinitis over the next 2-6 months. Unfortunately, the predictive value of CD4+ or CD8+ T cell responses for protection from ensuing retinitis was not statistically significant. However, there was a trend towards differences in the memory phenotype of patients that developed retinitis versus controls.
Maecker, H. T., J. Hassler, J. K. Payne, A. Summers, K. Comatas, M. Ghanayem, M. A. Morse, T. M. Clay, H. K. Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino, C. Delarosa, and M. L. Disis. 2008. Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC immunology 9:9. Link to full text.
This is the latest, and probably last paper to result from our Immune Monitoring Consortium. It attempts to define the precision and linearity of ELISPOT, ICS, and tetramer staining, using CMV peptides. Not surprisingly, the simple tetramer assay did very well, but ICS was not far behind. ELISPOT, while slightly more variable, could also be made to perform with reasonable precision and linearity, at least in a donor with a relatively high response.
Rolland, M., D. Heckerman, W. Deng, C. M. Rousseau, H. Coovadia, K. Bishop, P. J. Goulder, B. D. Walker, C. Brander, and J. I. Mullins. 2008. Broad and Gag-Biased HIV-1 Epitope Repertoires Are Associated with Lower Viral Loads. PLoS ONE 3:e1424. Link to full text.
The latest paper to suggest that broad and Gag-based T cell responses tend to be protective in lowering viral loads in HIV patients. While a tour de force in terms of its comprehensive analysis of a large number of patients, this paper also shows the complexity of trying to predict protection by measuring T cell responses alone. It's clear that a T cell response to Gag is not enough to predict viral load on a patient-by-patient basis.
See also the paper by Emu et al. Seoane, E., S. Resino, S. Moreno, J. C. de Quiros, A. Moreno, R. Rubio, J. Gonzalez-Garcia, J. R. Arribas, F. Pulido, and M. A. Munoz-Fernandez. 2008. Immunological predictors of CD4+ T cell decline in antiretroviral treatment interruptions. BMC infectious diseases 8:20. Link to full text.
When trying to predict how well patients fare upon ARV treatment interruption, these authors identify proliferative responses and IL-4 production to PPD stimulation as positive and negative predictors, respectively.
Soares, A. P., T. J. Scriba, S. Joseph, R. Harbacheuski, R. A. Murray, S. J. Gelderbloem, A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, and W. A. Hanekom. 2008. Bacillus calmette-guerin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles. J Immunol 180:3569-3577. Link to PubMed abstract.
This paper gives a bit more detail about the phenotypic and functional complexity of BCG-responsive T cells in vaccinated infants. Of note, IL-2-expressing T cells do not always co-express IFNg, as is generally the case with CMV-responsive T cells. And the predominant phenotype, while effector-like, is not terminally differentiated as is the case with CMV.
Oct-Dec 2007:
Borowski, A. B., A. C. Boesteanu, Y. M. Mueller, C. Carafides, D. J. Topham, J. D. Altman, S. R. Jennings, and P. D. Katsikis. 2007. Memory CD8+ T cells require CD28 costimulation. J Immunol 179:6494-6503 Link to PubMed abstract A report from Peter Katsikis’s laboratory challenging the current paradigm on the requirement of CD28 costimulation by memory CD8 T cells. The data using influenza A and HSV models show that following infection in vivo memory CD8 T cells require CD28 costimulation during reactivation to reach maximal expansion and effective pathogen clearance. These findings suggest that vaccine strategies should include ways to stimulate CD28 for effective secondary immune response. Bos, R., S. van Duikeren, T. van Hall, M. M. Lauwen, M. Parrington, N. L. Berinstein, B. McNeil, C. J. Melief, J. S. Verbeek, S. H. van der Burg, and R. Offringa. 2007. Characterization of antigen-specific immune responses induced by canarypox virus vaccines. J Immunol 179:6115-6122 Link to PubMed abstract For those working in ALVAC-based vaccine research, this report shows that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine. Garrison, K. E., R. B. Jones, D. A. Meiklejohn, N. Anwar, L. C. Ndhlovu, J. M. Chapman, A. L. Erickson, A. Agrawal, G. Spotts, F. M. Hecht, S. Rakoff-Nahoum, J. Lenz, M. A. Ostrowski, and D. F. Nixon. 2007. T cell responses to human endogenous retroviruses in HIV-1 infection. PLoS pathogens 3:e165 Link to PubMed abstract An interesting approach to control HIV infection reported by Doug Nixon’s group. They demonstrate that T cells respond to human endogenous retroviruses (HERV) when a person is infected with HIV. The T cells responding to HERV share characteristics (terminally differentiated phenotype) with T cells that effectively control CMV. T cells responding to HERV can also kill target cells carrying HERV protein. For some HIV-positive people, the strength of their response against HERV is related to having a lower HIV viral load. The authors propose that if T cells that recognize HERV can stably target HIV-infected cells, they could be an important factor in controlling HIV infection. Kaiko, G. E., J. C. Horvat, K. W. Beagley, and P. M. Hansbro. 2007. Immunological decision-making: how does the immune system decide to mount a helper T-cell response? Immunology Link to PubMed abstract A nice review article discussing many different pathways and key elements that act synergistically, antagonistically and through positive feedback loops to activate a Th1, Th2, or Th17 immune response. Sauce, D., J. R. Almeida, M. Larsen, L. Haro, B. Autran, G. J. Freeman, and V. Appay. 2007. PD-1 expression on human CD8 T cells depends on both state of differentiation and activation status. AIDS (London, England) 21:2005-2013 Link to PubMed abstract One more report on PD-1 expression by Victor Appay’s group trying to understand the detailed expression pattern of PD-1 on HIV-specific CD8 cells. These data show significant differences in PD-1 expression between the distinct CD8 T-cell subsets along the pathway of CD8 T-cell differentiation. Seresini, S., M. Origoni, F. Lillo, L. Caputo, A. M. Paganoni, S. Vantini, R. Longhi, G. Taccagni, A. Ferrari, C. Doglioni, P. Secchi, and M. P. Protti. 2007. IFN-gamma produced by human papilloma virus-18 E6-specific CD4+ T cells predicts the clinical outcome after surgery in patients with high-grade cervical lesions. J Immunol 179:7176-7183 Link to PubMed abstract An important finding of this study is the identification of an immunologic parameter (i.e., the level of IFN-g) produced by HPV-18-specific CD4+ T cells) that could be applied to discriminate, at the time of surgery, between patients with favorable or unfavorable clinical outcome. It would be interesting to see if ICS assay would provide similar results in terms of frequency of IFNg+CD4+ T cells. Wherry, E. J., S. J. Ha, S. M. Kaech, W. N. Haining, S. Sarkar, V. Kalia, S. Subramaniam, J. N. Blattman, D. L. Barber, and R. Ahmed. 2007. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity27:670-684 Link to PubMed abstract A very good study from Rafi Ahmed’s lab showing that CD8+ T cell exhaustion and anergy are distinct processes. These studies identify a number of inhibitory, signaling, and metabolic pathways that might underlie the functional defects in these CD8+ T cells and might also represent novel targets for immunotherapy. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein, J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, J. C. Lecron, R. A. Kastelein, D. J. Cua, T. K. McClanahan, E. P. Bowman, and R. de Waal Malefyt. 2007. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nature immunology 8:950-957 Link to PubMed abstract A comprehensive study describing the differentiation and cytokine/chemokine profiles of human Th17 cells. These cells are identified in situ by expression of IL23-receptor and CD45RO. Analysis of cells from the human psoriasis lesions further extend the involvement of TH-17 cells in innate immunity to neutrophil-independent mechanisms of antimicrobial protection.
Aug-Sept 2007:
Almeida, J. R., D. A. Price, L. Papagno, Z. A. Arkoub, D. Sauce, E. Bornstein, T. E. Asher, A. Samri, A. Schnuriger, I. Theodorou, D. Costagliola, C. Rouzioux, H. Agut, A. G. Marcelin, D. Douek, B. Autran, and V. Appay. 2007. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med. Link to PubMed abstract Appay and colleagues address the question, “Why do HLA-B27-restricted responses to the KK10 epitope confer protection from HIV?” The answers could be multiple, as suggested in the title: KK10-specific cells have high avidity, polyfunctional cytokine production, and high clonal turnover. Dong, J., C. Ivascu, H. D. Chang, P. Wu, R. Angeli, L. Maggi, F. Eckhardt, L. Tykocinski, C. Haefliger, B. Mowes, J. Sieper, A. Radbruch, F. Annunziato, and A. Thiel. 2007. IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes. J Immunol 179:2389. Link to PubMed abstract The capacity to express IFNg is controlled epigenetically, by DNA hyppomethylation. Thiel and colleagues show that this is not the case for IL-10, and that memory for IL-10 production is lacking, when IL-10-secreting cells are isolated, cultured, and restimulated. D'Souza, M., A. P. Fontenot, D. G. Mack, C. Lozupone, S. Dillon, A. Meditz, C. C. Wilson, E. Connick, and B. E. Palmer. 2007. Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction. J Immunol 179:1979. Link to PubMed abstract More on the PD-1 story, this time focused on HIV-specific CD4+ T cells. Inokuma, M., C. dela Rosa, C. Schmitt, P. Haaland, J. Siebert, D. Petry, M. Tang, M. A. Suni, S. A. Ghanekar, D. Gladding, J. F. Dunne, V. C. Maino, M. L. Disis, and H. T. Maecker. 2007. Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature. J Immunol 179:2627. Link to PubMed abstract Our study of T cell responses to tumor antigens versus CMV and flu, in breast cancer patients. We show a bias away from IFNg and towards IL-2 production in the tumor antigen responses; and also, a predominantly CM phenotype, as opposed to the more heterogeneous phenotypes seen in the CMV response. Johnston, M. I., and A. S. Fauci. 2007. An HIV vaccine--evolving concepts. N Engl J Med 356:2073. Link to PubMed abstract Summary of current thoughts around HIV vaccine development: neutralizing antibodies and CTLs, mucosal and systemic immunity, etc. Also a list of current trials and novel approaches. Okoye, A., M. Meier-Schellersheim, J. M. Brenchley, S. I. Hagen, J. M. Walker, M. Rohankhedkar, R. Lum, J. B. Edgar, S. L. Planer, A. Legasse, A. W. Sylwester, M. Piatak, Jr., J. D. Lifson, V. C. Maino, D. L. Sodora, D. C. Douek, M. K. Axthelm, Z. Grossman, and L. J. Picker. 2007. Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection. J Exp Med 204:2171. Link to PubMed abstract The Picker lab examines the natural history of CD4+ CM and EM CD4+ T cells in primary and then chronic SIV infection. They suggest that loss of EM cells is a proximal correlate of simian AIDS, but that this loss is controlled by the rate of production of new CM T cells, that can give rise to EM cells. Schlaphoff, V., C. S. Klade, B. Jilma, S. B. Jelovcan, M. Cornberg, E. Tauber, M. P. Manns, and H. Wedemeyer. 2007. Functional and phenotypic characterization of peptide-vaccine-induced HCV-specific CD8+ T cells in healthy individuals and chronic hepatitis C patients. Vaccine 25:6793. Link to PubMed abstract This study looks at the response to HCV-peptide vaccination in healthy donors, and also the HCV and CMV response in chronic hepatitis C. I have some criticisms of the data on relative IFNg production by tetramer+ cells (done across two platforms, by dividing ELISPOT results by tetramer staining numbers), but in general there is some useful information here. Streitz, M., L. Tesfa, V. Yildirim, A. Yahyazadeh, T. Ulrichs, R. Lenkei, A. Quassem, G. Liebetrau, L. Nomura, H. Maecker, H. D. Volk, and F. Kern. 2007. Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis. PLoS ONE 2:e735. Link to free full-text A test to distinguish latent from active TB would be very useful, and this paper from Florian Kern’s group suggests that loss of CD27 expression on TB-responsive T cells may be a good correlate of active disease. Vescovini, R., C. Biasini, F. F. Fagnoni, A. R. Telera, L. Zanlari, M. Pedrazzoni, L. Bucci, D. Monti, M. C. Medici, C. Chezzi, C. Franceschi, and P. Sansoni. 2007. Massive Load of Functional Effector CD4+ and CD8+ T Cells against Cytomegalovirus in Very Old Subjects. J Immunol 179:4283. Link to PubMed abstract Nice data on the accumulation of CD4+ and CD8+ CMV-specific T cells in very old seropositive donors. The cells appear functional by production of IFNg, TNFa, and mobilization of CD107. But no data is shown on PD-1, CD57, or other possible markers of clonal exhaustion; nor was proliferative capacity in vitro analyzed. Ward, S., and A. Dalgleish. 2007. Therapeutic cancer vaccines. Vaccine. Link to PubMed abstract Editorial on the prospects for therapeutic vaccines to solid tumors. June-July 2007: Almeida, J. R., D. A. Price, L. Papagno, Z. A. Arkoub, D. Sauce, E. Bornstein, T. E. Asher, A. Samri, A. Schnuriger, I. Theodorou, D. Costagliola, C. Rouzioux, H. Agut, A. G. Marcelin, D. Douek, B. Autran, and V. Appay. 2007. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med. Link to PubMed abstract Appay and colleagues address the question, “Why do HLA-B27-restricted responses to the KK10 epitope confer protection from HIV?” The answers could be multiple, as suggested in the title: KK10-specific cells have high avidity, polyfunctional cytokine production, and high clonal turnover. Dong, J., C. Ivascu, H. D. Chang, P. Wu, R. Angeli, L. Maggi, F. Eckhardt, L. Tykocinski, C. Haefliger, B. Mowes, J. Sieper, A. Radbruch, F. Annunziato, and A. Thiel. 2007. IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes. J Immunol 179:2389. Link to PubMed abstract The capacity to express IFNg is controlled epigenetically, by DNA hyppomethylation. Thiel and colleagues show that this is not the case for IL-10, and that memory for IL-10 production is lacking, when IL-10-secreting cells are isolated, cultured, and restimulated. D'Souza, M., A. P. Fontenot, D. G. Mack, C. Lozupone, S. Dillon, A. Meditz, C. C. Wilson, E. Connick, and B. E. Palmer. 2007. Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction. J Immunol 179:1979. Link to PubMed abstract More on the PD-1 story, this time focused on HIV-specific CD4+ T cells. Inokuma, M., C. dela Rosa, C. Schmitt, P. Haaland, J. Siebert, D. Petry, M. Tang, M. A. Suni, S. A. Ghanekar, D. Gladding, J. F. Dunne, V. C. Maino, M. L. Disis, and H. T. Maecker. 2007. Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature. J Immunol 179:2627. Link to PubMed abstract Our study of T cell responses to tumor antigens versus CMV and flu, in breast cancer patients. We show a bias away from IFNg and towards IL-2 production in the tumor antigen responses; and also, a predominantly CM phenotype, as opposed to the more heterogeneous phenotypes seen in the CMV response. Johnston, M. I., and A. S. Fauci. 2007. An HIV vaccine--evolving concepts. N Engl J Med 356:2073. Link to PubMed abstract Summary of current thoughts around HIV vaccine development: neutralizing antibodies and CTLs, mucosal and systemic immunity, etc. Also a list of current trials and novel approaches. Okoye, A., M. Meier-Schellersheim, J. M. Brenchley, S. I. Hagen, J. M. Walker, M. Rohankhedkar, R. Lum, J. B. Edgar, S. L. Planer, A. Legasse, A. W. Sylwester, M. Piatak, Jr., J. D. Lifson, V. C. Maino, D. L. Sodora, D. C. Douek, M. K. Axthelm, Z. Grossman, and L. J. Picker. 2007. Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection. J Exp Med 204:2171. Link to PubMed abstract The Picker lab examines the natural history of CD4+ CM and EM CD4+ T cells in primary and then chronic SIV infection. They suggest that loss of EM cells is a proximal correlate of simian AIDS, but that this loss is controlled by the rate of production of new CM T cells, that can give rise to EM cells. Schlaphoff, V., C. S. Klade, B. Jilma, S. B. Jelovcan, M. Cornberg, E. Tauber, M. P. Manns, and H. Wedemeyer. 2007. Functional and phenotypic characterization of peptide-vaccine-induced HCV-specific CD8+ T cells in healthy individuals and chronic hepatitis C patients. Vaccine 25:6793. Link to PubMed abstract This study looks at the response to HCV-peptide vaccination in healthy donors, and also the HCV and CMV response in chronic hepatitis C. I have some criticisms of the data on relative IFNg production by tetramer+ cells (done across two platforms, by dividing ELISPOT results by tetramer staining numbers), but in general there is some useful information here. Streitz, M., L. Tesfa, V. Yildirim, A. Yahyazadeh, T. Ulrichs, R. Lenkei, A. Quassem, G. Liebetrau, L. Nomura, H. Maecker, H. D. Volk, and F. Kern. 2007. Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis. PLoS ONE 2:e735. Link to free full-text A test to distinguish latent from active TB would be very useful, and this paper from Florian Kern’s group suggests that loss of CD27 expression on TB-responsive T cells may be a good correlate of active disease. Vescovini, R., C. Biasini, F. F. Fagnoni, A. R. Telera, L. Zanlari, M. Pedrazzoni, L. Bucci, D. Monti, M. C. Medici, C. Chezzi, C. Franceschi, and P. Sansoni. 2007. Massive Load of Functional Effector CD4+ and CD8+ T Cells against Cytomegalovirus in Very Old Subjects. J Immunol 179:4283. Link to PubMed abstract Nice data on the accumulation of CD4+ and CD8+ CMV-specific T cells in very old seropositive donors. The cells appear functional by production of IFNg, TNFa, and mobilization of CD107. But no data is shown on PD-1, CD57, or other possible markers of clonal exhaustion; nor was proliferative capacity in vitro analyzed. Ward, S., and A. Dalgleish. 2007. Therapeutic cancer vaccines. Vaccine. Link to PubMed abstract Editorial on the prospects for therapeutic vaccines to solid tumors. April-May 2007: Bull, M., D. Lee, J. Stucky, Y. L. Chiu, A. Rubin, H. Horton, and M. J. McElrath. 2007. Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57. Link to PubMed abstract Maybe this is not a big surprise, but it is nice to see published data confirming that it's important to minimize the time between blood collection and PBMC isolation/cryopreservation, for cells used in functional assays. Although processing cells after more than 8 hours resulted in only a small decrease in viability, it had a greater effect on cell recovery and IFNg+ cells in ELISPOT. Cellerai, C., A. Harari, F. Vallelian, O. Boyman, and G. Pantaleo. 2007. Functional and phenotypic characterization of tetanus toxoid-specific human CD4+ T cells following re-immunization. Eur J Immunol 37:1129. Link to PubMed abstract Tetanus toxoid-specific CD4+ T cells, although rare in peripheral blood, can be monitored by ICS. Pantaleo's group shows that these cells are primarily IL-2 producing, CCR7+, CD127+ central memory-like T cells. Upon re-immunization, many of these cells acquire IFNg production and lose CCR7, CD127, and Bcl-2 expression, becoming effector memory cells. The population returns to a central memory-like state around 60 days post-immunization. Cohavy, O., and S. R. Targan. 2007. CD56 Marks an Effector T Cell Subset in the Human Intestine. J Immunol 178:5524. Link to PubMed abstract The gut has a high percentage of CD56+ T cells, and these appear to be effector cells with limited proliferative potential. Perhaps this is related to the poor reconstitution of mucosal T cells upoon HAART (See Mehandru et al below)? Ghanekar, S. A., S. Bhatia, J. J. Ruitenberg, C. D. Rosa, M. L. Disis, V. C. Maino, H. T. Maecker, and C. A. Waters. 2007. Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors. J Immune Based Ther Vaccines 5:7. Link to pdf There are lots of papers suggesting defects in T cells and APCs of cancer patients, but here we show that monocyte-derived dendritic cells (MDDC) generated from cancer patient PBMC are not very different from those derived from healthy donor PBMC. This is good news for the cancer DC vaccine makers. Also, there is some detriment to using cryopreserved rather than fresh PBMC for generating MDDC, but only for two markers tested (CD86 and COX-2). In general, cryopreserved PBMC from cancer patients can be used to generate phenotypically mature and functional DCs. Glomski, I. J., J. P. Corre, M. Mock, and P. L. Goossens. 2007. Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis. J Immunol 178:2646. Link to PubMed abstract In addition to neutralizing antibodies angainst anthrax protective Ag, a CD4+ T cell response, producing IFNg, is shown to be important in spore-induced anthax immunity in mice. Perhaps not too surprising, but another piece of evidence that monitoring of cellular immunity should be included in evaluation of new anthrax vaccines. Horton, H., E. P. Thomas, J. A. Stucky, I. Frank, Z. Moodie, Y. Huang, Y. L. Chiu, M. J. McElrath, and S. C. De Rosa. 2007. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 323:39. Link to PubMed abstract More methodology from the McElrath group, this time for 8-color ICS assays. They show a variety of validation data here, including: the importance of overnight culture after thawing and prior to cell stimulation (I agree!); usefulness of washing cells after the overnight rest (new to me!); and questionable usefulness of CD28+CD49d costimulation for peptide responses (I generally agree, though our data are a bit more positive in favor of using costimulation). They also examine peptide pool size more carefully than we ever did, deciding that <100 peptides is best to avoid declining responses. And they show some data on the use of a viability dye, which excludes some dim PE positive events that could otherwise add background to a PE cytokine measurement (still not a truly definitive endorsement of the need for these dyes, compared to, for example, other methods of dead cell exclusion). They do observe that CD4+CD8 co-gating can reduce non-specific staining by allowing exclusion of double-posiitve events (I agree as well). Finally, they show some nice data on assay linearity and precision, and the relationship of mean and CV. Overall, excellent stuff! Khan, N., D. Best, R. Bruton, L. Nayak, A. B. Rickinson, and P. A. Moss. 2007. T cell recognition patterns of immunodominant cytomegalovirus antigens in primary and persistent infection. J Immunol 178:4455. Link to PubMed abstract Here the acute and chronic CD8+ T cell response to CMV pp65 and IE-1 is mapped using IFNg ELISPOT, and then ICS. Some interesting though not unexpected findings include the continued increase of IE-1, but not pp65 responses, over time; the rapid emergence of effector memory cells and eventual dominance of CD45RA+ effector cells, especially among IE-1 specific CD8+ T cells. Mehandru, S., M. A. Poles, K. Tenner-Racz, P. Jean-Pierre, V. Manuelli, P. Lopez, A. Shet, A. Low, H. Mohri, D. Boden, P. Racz, and M. Markowitz. 2006. Lack of Mucosal Immune Reconstitution during Prolonged Treatment of Acute and Early HIV-1 Infection. PLoS Med 3:e484. Link to pdf Unfortunately, even early HAART treatment does not lead to complete reconstitution of T cells in the gut mucosa, even though peripheral T cell reconstitution is good. Millington, K. A., J. A. Innes, S. Hackforth, T. S. Hinks, J. J. Deeks, D. P. Dosanjh, V. Guyot-Revol, R. Gunatheesan, P. Klenerman, and A. Lalvani. 2007. Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load. J Immunol 178:5217. Link to PubMed abstract These authors propose that combined measurement of IFNg and IL-2 in ICS might prove useful in differentiating active from latent TB. Patients with active disease had TB-specific CD4+ T cells that made IFNg alone or IFNg+IL-2; while during and after treatment, a population of cells making IL-2 only appeared. It is suggested that this could be a "functional signature" of disease status. Smith, J. G., H. R. Joseph, T. Green, J. A. Field, M. Wooters, R. M. Kaufhold, J. Antonello, and M. J. Caulfield. 2007. Establishing Acceptance Criteria for Cell-Mediated-Immunity Assays Using Frozen Peripheral Blood Mononuclear Cells Stored under Optimal and Suboptimal Conditions. Clin Vaccine Immunol 14:527. Link to PubMed abstract This is a much-needed set of data examining the best way to set a cutoff of acceptability for cryopreserved and thawed PBMC that are to be used for a functional assay (in this case ELISPOT). It's remarkable because of the number of samples studied, the comparison of several different assays for acceptability, and the fact that very clear data emerged to differentiate optimally stored versus suboptimally stored samples (stored at -20C or cycled between -70 and -130C). The winning assay turned out to be apoptosis (annexin V + 7-AAD staining), where a cutoff of <18% apoptotic cells was best able to separate samples responsive to peptide or VZV antigen stimulation. Very useful information for clinical trial designers!
Feb-Mar 2007:
Azzoni, L., J. Chehimi, L. Zhou, A. S. Foulkes, R. June, V. C. Maino, A. Landay, C. Rinaldo, L. P. Jacobson, and L. J. Montaner. 2007. Early and delayed benefits of HIV-1 suppression: timeline of recovery of innate immunity effector cells. Aids 21:293. Link to PubMed abstract NK cells and DC's are followed during viremia and after onset of ART in 66 HIV patients. Both short-term and long-term recovery of specific populations are seen. Doolan, D. L., D. A. Freilich, G. T. Brice, T. H. Burgess, M. P. Berzins, R. L. Bull, N. L. Graber, J. L. Dabbs, L. L. Shatney, D. L. Blazes, L. M. Bebris, M. F. Malone, J. F. Eisold, A. J. Mateczun, and G. J. Martin. 2007. The US capitol bioterrorism anthrax exposures: clinical epidemiological and immunological characteristics. J Infect Dis 195:174. Link to PubMed abstract The immunological data here is highly summarized (bar graphs of aggregate responses from various groups, with no error bars), but the subject is certainly interesting. And the conclusion, that CMI may occur at subclinical infection levels, is a provocative one. Gary Brice tells me that a more detailed paper on the cellular immunology is forthcoming, so we'll be looking for that. Khader, S. A., G. K. Bell, J. E. Pearl, J. J. Fountain, J. Rangel-Moreno, G. E. Cilley, F. Shen, S. M. Eaton, S. L. Gaffen, S. L. Swain, R. M. Locksley, L. Haynes, T. D. Randall, and A. M. Cooper. 2007. IL-23 and IL-17 in the establishment of protective pulmonary CD4(+) T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 8:369. Link to PubMed abstract Using intracellular staining of IL-17 versus IFNg in mouse T cells, these authors show that these are mostly mutually exclusive populations responding to mouse TB. Something to look at in human T cells.... Kierstead, L. S., S. Dubey, B. Meyer, T. W. Tobery, R. Mogg, V. R. Fernandez, R. Long, L. Guan, C. Gaunt, K. Collins, K. J. Sykes, D. V. Mehrotra, N. Chirmule, J. W. Shiver, and D. R. Casimiro. 2007. Enhanced rates and magnitude of immune responses detected against an hiv vaccine: effect of using an optimized process for isolating PBMC. AIDS Res Hum Retroviruses 23:86. Link to PubMed abstract It may seem obvious that processing blood within 12 hours is important for maximizing cell function, but these authors show this in the context of ELISPOT and an HIV vaccine trial, among other findings. Liu, J., and M. Roederer. 2007. Differential susceptibility of leukocyte subsets to cytotoxic T cell killing: implications for HIV immunopathogenesis. Cytometry A 71:94. Link to PubMed abstract Using a flow-based cytotoxicity assay, the Roederer lab finds that different subsets of T cells have different sensitivities to peptide-based CTL killing. Perfetto, S. P., and M. Roederer. 2007. Increased immunofluorescence sensitivity using 532 nm laser excitation. Cytometry A 71:73. Link to PubMed abstract As we've heard, and experienced on our own cytometer, now here is the data published: green laser excitation of PE and PE tandems yields greater resolution sensitvity than blue laser excitation. Our own unpublished data would suggest that even a 100 mW blue laser doesn't give as much of a sensitivity boost as a green laser. While the increased sensitivity may not seem like much on its own, when doing multiparameter work with several PE tandems, the benefits add up. Pira, G. L., F. Kern, J. Gratama, M. Roederer, and F. Manca. 2007. Measurement of antigen specific immune responses: 2006 update. Cytometry B Clin Cytom 72:77. Link to PubMed abstract Review of the MASIR meeting in Santorini, Greece last June. Sharpe, A. H., E. J. Wherry, R. Ahmed, and G. J. Freeman. 2007. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol 8:239. Link to PubMed abstract A review of what's happening in the PD-1/PD-1 ligand field, including in HIV, HCV, and autoimmune disease. Stubbe, M., N. Vanderheyde, M. Goldman, and A. Marchant. 2006. Antigen-specific central memory CD4+ T lymphocytes produce multiple cytokines and proliferate in vivo in humans. J Immunol 177:8185. Link to PubMed abstract These authors track the CD4+ T cell response to HepB immunization, using CD154, cytokines, Ki67, and memory/effector markers. They find that CCR7+ and CCR7- cells result from immunization, that both of these populations make IFNg and IL-2, and that some of the CCR7+ cells can be proliferating (Ki67+) even several years after immunization. Tilton, J. C., M. R. Luskin, A. J. Johnson, M. Manion, C. W. Hallahan, J. A. Metcalf, M. McLaughlin, R. T. Davey, Jr., and M. Connors. 2007. Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load. J Virol 81:2713. Link to PubMed abstract This paper nails down the relationship of viremia, IL-2 production, and proliferative capacity of HIV-specific CD4+ T cells. Nice work, as we've come to expect from this group. Dec 2006-Jan 2007: Abrams, B., and T. Dubrovsky. 2006. Quantum Dots in Flow Cytometry. In Quantum Dots: Applications in Biology, Vol. 374. M. Bruchez, and C. Hotz, eds. Human Press, Totowa, NJ, p. 185. Link to publisher's abstract A bit of chemistry and some protocol considerations when using Q-dots in multicolor flow cytometry, from our protein chemists at BD. Boyer, J. D., P. C. Maciag, R. Parkinson, L. Wu, M. G. Lewis, D. B. Weiner, and Y. Paterson. 2006. Rhesus macaques with high levels of vaccine induced IFN-gamma producing cells better control viral set-point following challenge with SIV239. Vaccine 24:4498. Link to PubMed abstract We no longer expect IFNg ELISPOT responses to correlate with much in terms of clinical prognosis. However, Boyer et al. find a weak but significant correlation of pre-challenge ELISPOT results with viral set-point (week 10-14 viral load) in vaccinated and SIV-challenged macaques. There was no correlation with peak viral load (week 2), or with viral load at a late time point (week 19); nor were post-challenge ELISPOT results correlated with viral load at week 14. Brenchley, J. M., D. A. Price, T. W. Schacker, T. E. Asher, G. Silvestri, S. Rao, Z. Kazzaz, E. Bornstein, O. Lambotte, D. Altmann, B. R. Blazar, B. Rodriguez, L. Teixeira-Johnson, A. Landay, J. N. Martin, F. M. Hecht, L. J. Picker, M. M. Lederman, S. G. Deeks, and D. C. Douek. 2006. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12:1365. Link to PubMed abstract The ability of the gut to exclude microorganisms can deteriorate in certain disease states, and chronic HIV infection is one of them. The resultant immune stimulation is a very likely explanation for systemic CD8+ T cell activation observed in chronic HIV infection, as this paper nicely shows. Chung, C., W. Lee, J. T. Loffredo, B. Burwitz, T. C. Friedrich, J. P. Giraldo Vela, G. Napoe, E. G. Rakasz, N. A. Wilson, D. B. Allison, and D. I. Watkins. 2007. Not all cytokine-producing CD8+ T cells suppress simian immunodeficiency virus replication. J Virol 81:1517. Link to PubMed abstract David Watkins' group uses an in vitro viral suppression assay (VSA) on cloned SIV-specific CD8+ T cells. They show that clones specific to the same epitope vary in suppression activity, and that suppression does not always correlate with cytokine production (although there is a positive correlation for IFNg). Loffredo, J. T., B. J. Burwitz, E. G. Rakasz, S. P. Spencer, J. J. Stephany, J. P. Giraldo Vela, S. R. Martin, J. Reed, S. M. Piaskowski, J. Furlott, K. L. Weisgrau, D. S. Rodrigues, T. Soma, G. Napoe, T. C. Friedrich, N. A. Wilson, E. G. Kallas, and D. I. Watkins. 2006. SIV-specific CD8+ T cell antiviral efficacy is unrelated to epitope specificity and abrogated by viral escape. J Virol. Link to PubMed abstract Very similar to the Chung et al. paper, this one uses the viral suppression assay (VSA) to compare the suppressive activity of cloned SIV-specific CD8+ T cells directed against epitopes from different proteins. They see no correlation of suppressive activity and early vs. late expression of the target protein. And they find that a clone that fails to suppress a variant virus can still make cytokines against that variant. Also, in contrast to the previous paper, this one shows that not all clones from elite controllers have good suppressive activity. In addition, they see no correlation of suppression and disease stage or viral load; so while they conclude that the VSA is superior to ELISPOT or ICS, it's not clear that VSA will predict clinical outcome any better than ELISPOT or ICS--and because it involves cloning T cells and an 8-day culture period, it's both impractical and indirect compared to ex vivo assays. Deviren, G., K. Gupta, M. E. Paulaitis, and J. P. Schneck. 2007. Detection of antigen-specific T cells on p/MHC microarrays. J Mol Recognit 20:32. Link to PubMed abstract A simple cell-binding assay, using peptide-MHC dimers spotted on glass slides, was first described by Mark Davis' group, and is explored in more detail here. Horton, H., I. Frank, R. Baydo, E. Jalbert, J. Penn, S. Wilson, J. P. McNevin, M. D. McSweyn, D. Lee, Y. Huang, S. C. De Rosa, and M. J. McElrath. 2006. Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection. J Immunol 177:7406. Link to PubMed abstract HIV-specific CD8+ T cell proliferative responses to HLA-B27 and B57 alleles are well-preserved even in chronic infection; while those to other epitopes lose their proliferative capacity with chronic infection. Kiepiela, P., K. Ngumbela, C. Thobakgale, D. Ramduth, I. Honeyborne, E. Moodley, S. Reddy, C. de Pierres, Z. Mncube, N. Mkhwanazi, K. Bishop, M. van der Stok, K. Nair, N. Khan, H. Crawford, R. Payne, A. Leslie, J. Prado, A. Prendergast, J. Frater, N. McCarthy, C. Brander, G. H. Learn, D. Nickle, C. Rousseau, H. Coovadia, J. I. Mullins, D. Heckerman, B. D. Walker, and P. Goulder. 2007. CD8(+) T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 13:46. Link to PubMed abstract You expect lots of patients in a Philip Goulder study, and this one delivers, with a cohort of 578 untreated HIV-infected individuals. Their CD8+ T cell responses were mapped. Breadth of Gag-specific responses was associated with decreasing viremia, while breadth of Env-specific responses was associated with increasing viremia. This was independent of HLA type. Kvale, E. O., Y. Floisand, F. Lund-Johansen, H. Rollag, L. Farkas, S. Ghanekar, P. Brandtzaeg, F. L. Jahnsen, and J. Olweus. 2006. Plasmacytoid DCs regulate recall responses by rapid induction of IL-10 in memory T cells. Blood. Link to PubMed abstract Plasmacytoid DC's (CD123+) are nicely differentiated from myeloid DC's (CD11c+) in their ability to induce IL-10 (versus IFNg) production from CD4+ memory T cells. Letvin, N. L. 2006. Progress and obstacles in the development of an AIDS vaccine. Nat Rev Immunol 6:930. Link to PubMed abstract A concise summary of where we are in the search for an HIV vaccine. Petrausch, U., D. Haley, W. Miller, K. Floyd, W. J. Urba, and E. Walker. 2006. Polychromatic flow cytometry: a rapid method for the reduction and analysis of complex multiparameter data. Cytometry A 69:1162. Link to PubMed abstract This paper deals with the informatics of polychromatic flow cytometry, using tools in the Winlist analysis software. Like Betts et al. (Blood 107:4781, 2006) and Nomura et al. (AIDS Res Ther 3:18, 2006), these authors create from their data a set of non-overlapping phenotypic cell subsets (coincidently, 32 subsets in all three cases), which are then compared across groups of samples. The present authors also do cluster analysis of the samples and display heat-maps of these results. Tobery, T. W., S. A. Dubey, K. Anderson, D. C. Freed, K. S. Cox, J. Lin, M. T. Prokop, K. J. Sykes, R. Mogg, D. V. Mehrotra, T. M. Fu, D. R. Casimiro, and J. W. Shiver. 2006. A comparison of standard immunogenicity assays for monitoring HIV type 1 gag-specific T cell responses in Ad5 HIV Type 1 gag vaccinated human subjects. AIDS Res Hum Retroviruses 22:1081. Link to PubMed abstract I hope you can stand one more assay-comparison paper. Tobery et al. compare ELISPOT, ICS, tetramer, and 51-Cr release assays in HIV gag-vaccinated subjects. Like Maecker et al. (BMC Immunology 6:17, 2005), the Merck group finds slightly higher sensitivity for ELISPOT compared to ICS (although this depends upon positive/negative cutoff criteria for each assay). They also find the best correlation between tetramer and ICS, with weaker correlation of ELISPOT and ICS. Tu, W., L. Potena, P. Stepick-Biek, L. Liu, K. Y. Dionis, H. Luikart, W. F. Fearon, T. H. Holmes, C. Chin, J. P. Cooke, H. A. Valantine, E. S. Mocarski, and D. B. Lewis. 2006. T-cell immunity to subclinical cytomegalovirus infection reduces cardiac allograft disease.Circulation 114:1608. Link to PubMed abstract CMV infection increases the chance of cardiac allograft rejection. Here it is shown that an early CD4+ T cell response to CMV (within one month of transplant) protects from both CMV viral load and acute rejection, as well as related adverse effects. Younes, S. A., L. Trautmann, B. Yassine-Diab, L. H. Kalfayan, A. E. Kernaleguen, T. O. Cameron, R. Boulassel, L. J. Stern, J. P. Routy, Z. Grossman, A. R. Dumont, and R. P. Sekaly. 2007. The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells. J Immunol 178:788. Link to PubMed abstract The Sekaly group nicely demonstrates that the time of HAART initiation correlates with the magnitude and breadth of proliferating HIV-specific CD4+ T cells. Too short of a time of viral exposure prior to HAART results in insufficient priming; while too long of a time results in depletion of responses. Zhang, J. Y., Z. Zhang, X. Wang, J. L. Fu, J. Yao, Y. Jiao, L. Chen, H. Zhang, J. Wei, L. Jin, M. Shi, G. F. Gao, H. Wu, and F. S. Wang. 2007. PD-1 upregulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors, but not in long-term non-progressors. Blood. Link to PubMed abstract Another PD-1 paper, confirming findings from the previously published studies, and extending them to a good-sized cohort of progressors versus non-progressors. Oct-Nov 2006: Horton, H., and S. C. De Rosa. 2006. Does preserving memory correlate with surviving HIV?Trends Microbiol. Link to PubMed abstract O.k., I haven't read this yet because I'm travelling and can't get the full-text pdf, but as a perspective on the HIV central memory debate, it's sure to be interesting. Cavenaugh, J. S., P. Snell, D. Jeffries, P. A. Waight, and S. J. McConkey. 2006. A relational database for management of flow cytometry and ELISpot clinical trial data. Cytometry B Clin Cytom. Link to PubMed abstract If you know a bit more about Microsoft Access than I do, you can almost certainly follow these guidelines to create a database for your clinical trial ELISPOT and/or flow cytometry results, along with other related information. What the authors don't do (as far as I can tell) is to link the actual FCS files to the database, so that one could search for a set of files, then download and re-analyze them. I think that such linking would be possible with Access, however. But the ability to seamlessly view, regate, and then update the data is still not here. Hahne, F., D. Arlt, M. Sauermann, M. Majety, A. Poustka, S. Wiemann, and W. Huber. 2006. Statistical methods and software for the analysis of high throughput reverse genetic assays using flow cytometry readouts. Genome Biol 7:R77. Link to PubMed abstract This is how a statistician might think about simple flow cytometry data, and it approaches the informatics problem from a very different angle than the previous paper. The open-source software described here is intended to statistically analyze large numbers of files, then display the results in ways that would simplify high-throughput screening assays. Some interesting stuff, but I fear that non-experts will have a difficult time using these tools. Perfetto, S. P., D. Ambrozak, R. Nguyen, P. Chattopadhyay, and M. Roederer. 2006. Quality assurance for polychromatic flow cytometry. Nat Protocols 1:1522. Link to online abstract Concise guidance on what needs to be done in terms of quality control for polychromatic flow cytometery is certainly needed, and this protocol supplies just that. Unfortunately, there is a lot of advice here that the average user probably never needs to know...much like instructing a neighborhood mechanic how to tune race cars. Also, from a company perspective, I would submit that much of the initial instrument validation should be handled at the factory and by the installing technician. I suppose the hope is that awareness of the issues raised here will result in better manufacturer QC and simplified (and automated) user QC routines in the future. Lamoreaux, L. L., M. Roederer, and R. Koup. 2006. Intracellular cytokine optimization and standard operating procedure. Nat Protocols 1:1507. Link to online abstract This protocol lays out some steps the authors consider important in optimizing multiparameter ICS assays, and uses the 5-function, 12-color VRC panel as an example of an optimized assay. The advice is good, albeit conservative: as we gain experience and success with certain antibodies and panels, it may not be necessary to titrate every Ab to the degree described here. One picky detail: Why spin at 863 g? Besides being an odd number, this seems way too harsh for cells prior to fixation. Maybe a misprint.... Murray, R. A., N. Mansoor, R. Harbacheuski, J. Soler, V. Davids, A. Soares, A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, and W. A. Hanekom. 2006. Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8+ T cell response. J Immunol 177:5647. Link to PubMed abstract Here we see that BCG vaccination of newborns induces a CD8 as well as CD4 T cell response. The most intriguing finding, though, is that CD8 cells responding to BCG tend to either secrete IFNg or degranulate, but rarely do both. This "signature" is in contrast to that for CMV or HIV, where most IFNg expression is concordant with degranulation. Of course, I have no idea why this should be different in this case... Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette, M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, J. P. Routy, E. K. Haddad, and R. P. Sekaly. 2006. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12:1198. Link to PubMed abstract I should have included this last month as a complementary treatise on PD-1 as a marker of CD8 T cell dysfunction in HIV. Nice work by Rafick Sekaly's group. Aug-Sept 2006: 1. Altfeld, M., and T. M. Allen. 2006. Hitting HIV where it hurts: an alternative approach to HIV vaccine design. Trends Immunol. in press Link to PubMed abstract This review makes the argument for vaccination with highly selected epitopes of HIV, designed to elicit responses that control infection (because mutations in those epitopes are highly detrimental to viral fitness). The sad part is that such protective epitopes are largely unknown for the most common HLA types. So while making sure that an HLA-B57+ individual becomes a long-term nonprogressor might be easy, doing the same for an HLA-A2+ individual might be hard. 2. Horton, H., C. Havenar-Daughton, D. Lee, E. Moore, J. Cao, J. McNevin, T. Andrus, H. Zhu, A. Rubin, T. Zhu, C. Celum, and M. J. McElrath. 2006. Induction of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV vaccine trial participants who subsequently acquire HIV-1 infection. J Virol 80:9779. Link to PubMed abstract Breakthrough infections after vaccination afford an opportunity to see how a vaccine might be helping (or hurting) the control of viremia. In the breakthrough infections studied here, the disappointing finding is that responses to epitopes in the vaccine were not significantly higher than responses to epitopes that were not in the vaccine. On the other hand, there was no evidence for vaccination causing any detriment to the subsequent response to infection. 3. Picker, L. J., E. F. Reed-Inderbitzin, S. I. Hagen, J. B. Edgar, S. G. Hansen, A. Legasse, S. Planer, M. Piatak, Jr., J. D. Lifson, V. C. Maino, M. K. Axthelm, and F. Villinger. 2006. IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates.J Clin Invest 116:1514. Link to pdf IL-15 may be a uniquely important adjuvant to immune reconstitution under ART, as demonstrated by this study showing its selective effects on effector memory T cells. 4. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy, E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, Z. Mncube, J. Duraiswamy, B. Zhu, Q. Eichbaum, M. Altfeld, E. J. Wherry, H. M. Coovadia, P. J. Goulder, P. Klenerman, R. Ahmed, G. J. Freeman, and B. D. Walker. 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443:350. Link to PubMed abstract A beautiful example of moving immunology from mouse models to human disease, this study shows upregulation of PD-1 on HIV-specific T cells, and correlation of PD-1 levels with disease progression. The best part is that blockade of the PD-1 pathway may be a viable therapeutic option. 5. Nomura, L. E., B. Emu, R. Hoh, P. Haaland, S. G. Deeks, J. N. Martin, J. M. McCune, D. F. Nixon, and H. T. Maecker. 2006. IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells. AIDS Res Ther 3:18. Link to pdf We finally published this study, from which I've been presenting data for the last 2-3 years. It correlates effector CD8+ T cell differentiation and CD8+ IL-2 production, in HIV-specific cells of patients with progressive disease. This correlation suggests that perhaps IL-2 is required to fully drive CD8+ T cells to an effector phenotype (e.g., CD27-CD28-CD45RA+). When we first started the study, the 8-color ICS was pretty novel. I suppose it's a good sign for the field that now it seems much more routine. 6. Sun, Y., J. E. Schmitz, A. P. Buzby, B. R. Barker, S. S. Rao, L. Xu, Z. Y. Yang, J. R. Mascola, G. J. Nabel, and N. L. Letvin. 2006. Virus-specific cellular immune correlates of survival in vaccinated monkeys after simian immunodeficiency virus challenge. J Virol. in press Link to PubMed abstract The Letvin group continues to find correlates of protection in vaccinated monkeys following SIV challenge. While there is a tendency to simplify a bit the classification of memory T cells (central vs. effector memory), the results are clear: surviving monkeys mount a CD4 and CD8 response, their T cells are multifunctional (making at least 2 of the 3 cytokines measured), and they maintain a relatively early memory phenotype. No exhaustion here! 7. He, X. S., T. H. Holmes, C. Zhang, K. Mahmood, G. W. Kemble, D. B. Lewis, C. L. Dekker, H. B. Greenberg, and A. M. Arvin. 2006. Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines. J Virol. in press Link to PubMed abstract This comparison of live attenuated and inactived flu vaccines highlights the variable nature of human responses to influenza, both before and after vaccination. Interestingly, adult T cell responses were not significantly boosted, on average, by either vaccine; but live attenuated vaccine preferentially boosted responses in 5-9 year old children (who presumably have had less natural exposure than adults). 8. Maecker, H. T., and J. Trotter. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037. Link to PubMed abstract Readers of my reference updates probably know all this stuff, but here is a tutorial that students might find useful when thinking about how to set up a flow cytometer, what controls to include, and how to interpret their results. 9. Perfetto, S. P., P. K. Chattopadhyay, L. Lamoreaux, R. Nguyen, D. Ambrozak, R. A. Koup, and M. Roederer. 2006. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313:199. Link to PubMed abstract Amine-reactive dyes are the new EMA, that is, they are replacing EMA as a means to discriminate dead cells in samples that then get fixed and permeabilized (as for ICS). Here the VRC group shows how these dyes compare to EMA and Annexin V, how to compensate them, and how lack of viability gating can cause background/artifacts. The question remains: do you need viability gating for your samples? If you have a significant dead cell population, the answer is probably yes. While we've never seen the level of background and artifacts from dead cells that is shown here, it is of course better to be safe than sorry. 10. Chattopadhyay, P. K., D. A. Price, T. F. Harper, M. R. Betts, J. Yu, E. Gostick, S. P. Perfetto, P. Goepfert, R. A. Koup, S. C. De Rosa, M. P. Bruchez, and M. Roederer. 2006. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12:972. Link to PubMed abstract A Qdot tour-de-force! In addition to revealing the details of their system for detection of Qdots, our VRC friends show us with pretty colors how variable the phenotype of tetramer+ T cells can be. Now if we could just solve the stability problems of these reagents. July 2006: 1. Appay, V., C. Jandus, V. Voelter, S. Reynard, S. E. Coupland, D. Rimoldi, D. Lienard, P. Guillaume, A. M. Krieg, J. C. Cerottini, P. Romero, S. Leyvraz, N. Rufer, and D. E. Speiser. 2006. New Generation Vaccine Induces Effective Melanoma-Specific CD8+ T Cells in the Circulation but Not in the Tumor Site. J Immunol 177:1670. link to PubMed abstract Once again the name of Victor Appay graces the literature, this time in a tumor immunology setting. And as usual, it's important stuff: tumor-specific T cells induced by vaccination are functional in the circulation, but get functionally suppressed (apparently by regulatory T cells) at the tumor site. 2. Carrasco, J., D. Godelaine, A. Van Pel, T. Boon, and P. van der Bruggen. 2006. CD45RA on human CD8 T cells is sensitive to the time elapsed since the last antigenic stimulation.Blood. link to PubMed abstract I'm not sure what to think of this paper, which is potentially quite interesting. It suggests that CD45RA expression can be lost and later regained from "terminal effector" cells after antigen exposure. This runs counter to the traditional notion that CD45RA expression, at least on antigen-experienced cells, marks a population that is low in proliferation potential and "terminally differentiated". However, the data in this paper are based on T cell clones and in vitro proliferation, and I'm not sure how well that relates to what happens in vivo. 3. Herzenberg, L. A., J. Tung, W. A. Moore, L. A. Herzenberg, and D. R. Parks. 2006. Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7:681. link to PubMed abstract I'm perplexed, but I guess pleasantly so, that Nature Immunology would publish a tutorial paper on how to display flow cytometry data. I suppose it speaks to the importance of flow to the immunology community. And the paper makes some good points. If I might pick a very small bone, I would take issue with the heading "Collect, then compensate". With autocompensation in Diva, there is no drawback to compensating before collecting test samples, as the compensation can always be redone. The authors acknowledge this further down in that section, but I think it should change the overall message to "Do compensation via the software" rather than "Collect before compensating". But it's a small point in a very sensible paper. 4. Mahic, M., S. Yaqub, C. C. Johansson, K. Tasken, and E. M. Aandahl. 2006. FOXP3+CD4+CD25+ adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism. J Immunol 177:246. link to PubMed abstract I include this paper to alert you to the idea that cyclooxygenase 2 (COX-2) can be used as an intracellular staining marker of Treg activity. 5. Masopust, D., S. J. Ha, V. Vezys, and R. Ahmed. 2006. Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. J Immunol 177:831. link to PubMed abstract Multiple boosting tends to induce effector cells that go to non-lymphoid sites, as shown here in a mouse LCMV model. Beware of histogram overload in the figures. 6. Munks, M. W., K. S. Cho, A. K. Pinto, S. Sierro, P. Klenerman, and A. B. Hill. 2006. Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection. J Immunol 177:450. link to PubMed abstract Using the murine CMV model, this paper makes a small but important point: T cells responding to different viral proteins have different effector/memory phenotypes, which correspond to their expansion or contraction over time. Thinking beyond this single mouse strain, this implies that HLA type will influence the memory/effector phenotype of a response, by selecting the possible epitopes to which an individual can respond. 7. Rock, M. T., S. M. Yoder, T. R. Talbot, K. M. Edwards, and J. E. Crowe, Jr. 2006. Cellular Immune Responses to Diluted and Undiluted Aventis Pasteur Smallpox Vaccine. J Infect Dis 194:435. link to PubMed abstract I definitely like the rigor with which the Crowe group does multicolor flow. In the first figure, they demonstrate the precision of their assay using a Levy-Jennings plot. Beyond that, the science is good, too, albeit a simple message: diluted smallpox vaccine induces a similar quality and quantity of responding T cells as the full-strength vaccine. 8. Speiser, D. E., P. Baumgaertner, C. Barbey, V. Rubio-Godoy, A. Moulin, P. Corthesy, E. Devevre, P. Y. Dietrich, D. Rimoldi, D. Lienard, J. C. Cerottini, P. Romero, and N. Rufer. 2006. A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination. J Immunol 177:1338. link to PubMed abstract How much data can you generate from one patient? This study has to be close to a record. The authors chart the course of a spontaneously induced melan-A specific T cell clone in a melanoma patient. The clone is boosted by vaccinaton, and the disease stabilizes. Then the tumor loses melan-A expression, and the disease progresses despite the T cell response. It's a bit like an Aesop's fable, but with a somewhat depressing moral to the story. June 2006: 1. Duvall, M. G., A. Jaye, T. Dong, J. M. Brenchley, A. S. Alabi, D. J. Jeffries, M. van der Sande, T. O. Togun, S. J. McConkey, D. C. Douek, A. J. McMichael, H. C. Whittle, R. A. Koup, and S. L. Rowland-Jones. 2006. Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection. J Immunol 176:6973. link to PubMed abstract More on the importance of polyfunctional (IFNg+IL-2+) T cells, this time in the CD4 compartment: HIV-2-specific CD4+ T cells tend to proliferate and be polyfunctional, while HIV-1-specific T cells do not, even in asymptomatic subjects. 2. Letvin, N. L., J. R. Mascola, Y. Sun, D. A. Gorgone, A. P. Buzby, L. Xu, Z. Y. Yang, B. Chakrabarti, S. S. Rao, J. E. Schmitz, D. C. Montefiori, B. R. Barker, F. L. Bookstein, and G. J. Nabel. 2006. Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys. Science 312:1530. link to PubMed abstract Another way to say "polyfunctional" may be "central memory". Here Norm Letvin's group shows that reduced viremia and increased survival post-challenge are associated with preservation of HIV-specific CD4+ central memory cells (CD28+CD95+), in an SIV vaccine model. Best of all, these survivors could be predicted by the magnitude of the peak IFNg+ responses by ELISPOT or CD4+ ICS. 3. Jansen, C. A., D. van Baarle, and F. Miedema. 2006. HIV-specific CD4+ T cells and viremia: who's in control? Trends Immunol 27:119. link to PubMed abstract A review of the relationship between HIV-specific CD4+ T cell responses and viral load, which are thought to correlate up to a certain level of viremia, beyond which the correlation breaks down or becomes inverse. 4. Pantaleo, G., and A. Harari. 2006. Functional signatures in antiviral T-cell immunity for monitoring virus-associated diseases. Nat Rev Immunol 6:417. link to PubMed abstract A similar review to Jansen et al., but focusing more specifically on the cytokine signatures (IFNg and IL-2) of HIV-specific T cells versus those specific for other viruses which are either controlled or cleared after infection. 5. Munks, M. W., M. C. Gold, A. L. Zajac, C. M. Doom, C. S. Morello, D. H. Spector, and A. B. Hill. 2006. Genome-wide analysis reveals a highly diverse CD8 T cell response to murine cytomegalovirus. J Immunol 176:3760. link to PubMed abstract The C57BL/6 mouse CD8 T cell response to murine CMV is approximately as diverse as the human response to HCMV. A lot of work is summarized here, including epitope mapping, functional avidity, and immunodominance in different mouse strains. 6. Samri, A., C. Durier, A. Urrutia, I. Sanchez, H. Gahery-Segard, S. Imbart, M. Sinet, E. Tartour, J. P. Aboulker, B. Autran, and A. Venet. 2006. Evaluation of the interlaboratory concordance in quantification of human immunodeficiency virus-specific T cells with a gamma interferon enzyme-linked immunospot assay. Clin Vaccine Immunol 13:684. link to PubMed abstract More on ELISPOT reproducibility, from the ANRS ELISPOT Standardization Group. These four labs were able to reach an inter-lab CV of 18.7%, comparable to the results with ICS in a 16-lab study [Maecker et al., BMC Immunol 6:13 (2005)]. 7. Williams, M. A., A. J. Tyznik, and M. J. Bevan. 2006. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature 441:890. link to PubMed abstract My ability to comprehend the highly manipulated mouse systems in which studies like this are carried out are unfortunately limited. What I can understand is that this group has devised a system whereby lack of IL-2 signalling in CD8+ T cells causes normal primary responses, but essentially no secondary response to LCMV. This obviously underscores the importance of IL-2 signalling in expanding a primed CD8+ T cell population. May 2006: 1. Ahmadzadeh, M., and S. A. Rosenberg. 2006. IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients. Blood 107:2409. Link to pdf Perhaps not surprisingly, systemic IL-2 adminstration increases the frequency of regulatory T cells. This is not true for IL-7 (see Rosenberg et al., J Immuntherapy, 2006, 29: 313). 2. Hicks, A. M., G. Riedlinger, M. C. Willingham, M. A. Alexander-Miller, C. Von Kap-Herr, M. J. Pettenati, A. M. Sanders, H. M. Weir, W. Du, J. Kim, A. J. Simpson, L. J. Old, and Z. Cui. 2006. Transferable anticancer innate immunity in spontaneous regression/complete resistance mice. Proc Natl Acad Sci U S A 103:7753. Link to pdf This paper highlights the importance of innate immune cells (NK cells, macrophages, and neutrophils) in anti-cancer immunity, and the transferable nature of that immunity. 3. Jacobson, M. A., E. Sinclair, B. Bredt, L. Agrillo, D. Black, C. L. Epling, A. Carvidi, T. Ho, R. Bains, V. Girling, and S. P. Adler. 2006. Safety and immunogenicity of Towne cytomegalovirus vaccine with or without adjuvant recombinant interleukin-12. Vaccine. Link to PubMed abstract This paper establishes the type and level of T cell responses that can be expected from CMV vaccination, in the presence or absence of adjuvant IL-12. Some of the apparent IL-12 effects are not anticipated (e.g., decreased peak CD4 T cell proliferative responses to IE-1). While it doesn't make a compelling case for using IL-12, it suggests there might be some overall benefit. 4. Krutzik, P. O., and G. P. Nolan. 2006. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3:361. Link to PubMed abstract By using titrated combinations of one or more small-molecule dyes, one can differentially label populations of cells, for example giving cells in each well of a microtiter plate a specific combination of dyes (a "barcode"). Suppose this is done after differentially treating the wells in an assay such as a drug screen. The wells can then all be combined into a single sample, and read very quickly on a flow cytometer, with the different wells being "separated" again via gating on the labeling dyes during analysis. In essence, this allows for multiplexed flow cytometry by using one or more colors to separate discreet samples that have been combined for acquisition. 5. McElhaney, J. E., D. Xie, W. D. Hager, M. B. Barry, Y. Wang, A. Kleppinger, C. Ewen, K. P. Kane, and R. C. Bleackley. 2006. T cell responses are better correlates of vaccine protection in the elderly. J Immunol 176:6333. Link to PubMed abstract This is the kind of paper that a cellular immunologist likes to see. It gives evidence for the importance of monitoring T cell responses, as opposed to Ab titers, to establish influenza vaccine protection in elderly adults. Unfortunately the staining in Figure 5 (C&D) doesn't look that clean, with regard to IFNg and IL-10. I would certainly not expect such coordinate expression of these two cytokines, nor would I expect such high percentages of PBMC to be flu-specific. I would, however, expect brighter staining, at least for IFNg, than what is shown. But perhaps the ELISA data is more convincing. 6. Ruitenberg, J. J., C. B. Mulder, V. C. Maino, A. L. Landay, and S. A. Ghanekar. 2006. VACUTAINER(R) CPT TM and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV Seropositive Blood Samples. BMC Immunol 7:11. Link to pdf Shipping samples is a fact of life for large clinical trials, but the effect on functional assays is often worrisome. These authors show that Vacutainer CPT tubes preserve function as well as Ficoll separation, and thus provide a convenient means to ship PBMC. April 2006: Acierno, P. M., J. E. Schmitz, D. A. Gorgone, Y. Sun, S. Santra, M. S. Seaman, M. H. Newberg, J. R. Mascola, G. J. Nabel, D. Panicali, and N. L. Letvin. 2006. Preservation of Functional Virus-Specific Memory CD8+ T Lymphocytes in Vaccinated, Simian Human Immunodeficiency Virus-Infected Rhesus Monkeys. J Immunol 176:5338. Link to PubMed abstract SIV vaccines using a DNA prime and viral vector boost can protect monkeys from SIV disease. Here the Letvin group shows that vaccinated animals, upon challenge, tend to preserve a central memory T cell population of SIV-specific CD8+ T cells (tetramer+) that can make IFNg, as well as an effector memory population that also produces granzyme B. The stretch to other work might be a bit thin, but I would submit that this is consistent with the idea that a heterogeneous population of responsive cells, including both "early" and "late" maturation stages, is important for protection. Chang, J. J., F. Wightman, A. Bartholomeusz, A. Ayres, S. J. Kent, J. Sasadeusz, and S. R. Lewin. 2005. Reduced hepatitis B virus (HBV)-specific CD4+ T-cell responses in human immunodeficiency virus type 1-HBV-coinfected individuals receiving HBV-active antiretroviral therapy. J Virol 79:3038. Link to pdf I missed this paper last year, but thought it was worth including now for the nice demonstration (the first to my knowledge) of ICS responses to HBV, using 15-mer peptide pools. It is quite a tour de force. I apologize if this is old news to you. Druilhe, P., J. P. Sauzet, K. Sylla, and C. Roussilhon. 2006. Worms can alter T cell responses and induce regulatory T cells to experimental malaria vaccines. Vaccine. Link to PubMed abstract This is a letter to the editor, dealing with the subject of helminthic parasite infection and its effect on a heterologous (malarial) immune response. It shows some compelling data and lists other publications in this area. The bottom line: endemic parasites can reduce IFNg and increase IL-10 production to a heterologous antigen. This merits concern for worldwide vaccine efforts not only for malaria, but for HIV and other infections as well. Frahm, N., P. Kiepiela, S. Adams, C. H. Linde, H. S. Hewitt, K. Sango, M. E. Feeney, M. M. Addo, M. Lichterfeld, M. P. Lahaie, E. Pae, A. G. Wurcel, T. Roach, M. A. St John, M. Altfeld, F. M. Marincola, C. Moore, S. Mallal, M. Carrington, D. Heckerman, T. M. Allen, J. I. Mullins, B. T. Korber, P. J. Goulder, B. D. Walker, and C. Brander. 2006. Control of human immunodeficiency virus replication by cytotoxic T lymphocytes targeting subdominant epitopes. Nat Immunol 7:173. Link to PubMed abstract I tend to avoid much of the literature on HIV epitopes and escape, but I have to pay homage to Christian Brander and his colleagues. I'm not sure if it's fair to draw a ratio between the number of authors on the paper and the number of patients studied, but for this group, both numbers tend to be very high. Anyway, the point of this study is that subdominant epitope responses can make all the difference in terms of viral control, and therefore they deserve more focus from vaccine designers. I'll leave the details to your reading. Khurana, S., J. Needham, B. Mathieson, I. R. Rodriguez-Chavez, A. T. Catanzaro, R. T. Bailer, J. Kim, V. Polonis, D. A. Cooper, J. Guerin, M. L. Peterson, M. Gurwith, N. Nguyen, B. S. Graham, and H. Golding. 2006. Human immunodeficiency virus (HIV) vaccine trials: a novel assay for differential diagnosis of HIV infections in the face of vaccine-generated antibodies. J Virol 80:2092. Link to PubMed abstract Here's a paper about a topic that I think hasn't received enough attention: how to differentiate HIV seroconversion from a vaccine-generated antibody response. It's important for vaccine trial volunteers who need to avoid being falsely labeled as HIV+ based on serology, and it's also important for researchers to discriminate breakthrough infections in vaccine recipients. This study proposes an assay based on conserved but non-protected Ab epitopes that are not found in most current vaccine candidates. Wickelgren, I. 2006. Immunology. Targeting the tolls. Science 312:184. Link to PubMed abstract If you still don't think that Toll-like receptors are particularly important to the immune response, this little overview should change your mind. There is some historical review, a table of receptors and what they see, and a focus on targeting these molecules with drugs. March 2006: 1. Asquith, B., C. T. Edwards, M. Lipsitch, and A. R. McLean. 2006. Inefficient Cytotoxic T Lymphocyte-Mediated Killing of HIV-1-Infected Cells In Vivo. PLoS Biol 4:e90. Link to pdf I can't critique these mathematical model papers, but I can summarize what I think they say: in this case, that CTLs kill a lot of HIV-infected cells, but they're not the major source of infected cell death in vivo. Don't ask me how they could conclude this, but there are some interesting ideas here. 2. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682. Link to PubMed abstract Exhaustion of CD8+ T cells is a concept pioneered by Rafi Ahmed's group. It is a common phenomenon in chronic viral infection, characterized by progressive lack of T cell function. This paper is particularly interesting in that it suggests a way to rescue cells from exhaustion, via blockade of the PD-1 receptor that is selectively upregulated on exhausted T cells. If the human situation is analogous, this would seem to be a therapy highly suited to diseases like HIV. 3. Ida, J. A., N. Shrestha, S. Desai, S. Pahwa, W. A. Hanekom, and P. A. Haslett. 2006. A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation. J Immunol Methods 310:86. Link to PubMed abstract Tips on doing functional cytokine studies of dendritic cell subsets, using TLR agonists in whole blood. 4. Jansen, C. A., I. M. De Cuyper, B. Hooibrink, A. K. van der Bij, D. van Baarle, and F. Miedema. 2006. Prognostic value of HIV-1 Gag-specific CD4+ T-cell responses for progression to AIDS analyzed in a prospective cohort study. Blood 107:1427. Link to PubMed abstract This paper encompases both a comparison study of 7 progressors and 6 long-term non-progressors, as well as a prospective study of 96 HIV seroconvertors. The aim was to determine whether HIV-specific CD4+ T cells producing IFNg, IL-2, or IFNg+IL-2 were predictive of disease progression. While the loss of IL-2 and IFNg-producing CD4+ T cells specific for HIV was seen late in progressive disease, there was no prognostic value to the magnitude of these T cell populations early after seroconversion. The authors draw a "virologist's" conclusion: that viral load determines the T cell response to HIV, and not the other way around. Maybe an overstatement, but interesting. 5. Kaufmann, S. H. 2005. Recent findings in immunology give tuberculosis vaccines a new boost. Trends Immunol 26:660. Link to PubMed abstract A review of what's needed for a more successful TB vaccine. 6. O'Connor D, H., and D. R. Burton. 2006. Immune responses and HIV: a little order from the chaos. J Exp Med 203:501. Link to PubMed abstract This perspective reviews two studies of HIV-infected twins and their Ab and T cell responses. I didn't have the patience to read the two studies, so I just read this. It seems that initially shared CTL responses tend to diverge by late-stage infection.