Biomedical subjects

Kaja Murali-Krishna

Publications and source records attributed to Kaja Murali-Krishna.

15 recordsLinked to original sources

PubMed2006

Quantitating the magnitude of the lymphocytic choriomeningitis virus-specific CD8 T-cell response: it is even bigger than we thought.

Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide > or =15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.

Animals↗

PubMed2006

Innate inflammatory signals induced by various pathogens differentially dictate the IFN-I dependence of CD8 T cells for clonal expansion and memory formation.

Type-I IFNs (IFN-I) provide direct survival signals to T cells during Ag-driven proliferation. Because IFN-I production differs depending on the pathogen, we assessed CD8 T cell requirement for direct IFN-I signals during responses to vaccinia virus (VV), vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), and Listeria monocytogenes (LM) immunizations in vivo. IFN-I-receptor-deficient (IFN-IR(o)) CD8 T cells expanded 3- to 5-fold less and formed a diminished memory pool compared with wild-type (WT) CD8 T cells in response to VV, VSV, or LM. WT CD8 T cells expanded more robustly in response to LCMV-encoded Ags than to Ags encoded by the other three pathogens, and under these conditions the lack of direct IFN-I signals inhibited their expansion by approximately 100-fold. To test whether the high antigenic-load provided by LCMV caused greater expansion and greater IFN-I dependency, we primed WT and IFN-IR(o) OVA-specific OT-1 CD8 T cells with a fixed-number of OVA-peptide-pulsed dendritic cells along with adjuvant effect provided by LCMV, VV, VSV, or LM. Both WT and IFN-IR(o) OT-1 cells were recruited, proliferated, and differentiated into effectors in all the four cases. However, WT OT-1 cells expanded similarly in all four cases. IFN-IR(o) OT-1 cells expanded approximately 20-fold less than the WT OT-1 CD8 T cells when LCMV was used as adjuvant, whereas their expansion was affected only marginally when VV, VSV, or LM were used as adjuvants. Thus, innate/inflammatory signals induced by different pathogens contribute to CD8 T cell expansion and memory formation via distinct levels of IFN-I dependence.

Animals↗

PubMed2006

Rapid demethylation of the IFN-gamma gene occurs in memory but not naive CD8 T cells.

DNA methylation is an epigenetic mechanism of gene regulation. We have determined that specific modifications in DNA methylation at the IFN-gamma locus occur during memory CD8 T cell differentiation in vivo. Expression of the antiviral cytokine IFN-gamma in CD8 T cells is highly developmental stage specific. Most naive cells must divide before they express IFN-gamma, while memory cells vigorously express IFN-gamma before cell division. Ag-specific CD8 T cells were obtained during viral infection of mice and examined directly ex vivo. Naive cells had an IFN-gamma locus with extensive methylation at three specific CpG sites. An inhibitor of methylation increased the amount of IFN-gamma in naive cells, indicating that methylation contributes to the slow and meager production of IFN-gamma. Effectors were unmethylated and produced large amounts of IFN-gamma. Interestingly, while memory cells were also able to produce large amounts of IFN-gamma, the gene was partially methylated at the three CpG sites. Within 5 h of antigenic stimulation, however, the gene was rapidly demethylated in memory cells. This was independent of DNA synthesis and cell division, suggesting a yet unidentified demethylase. Rapid demethylation of the IFN-gamma promoter by an enzymatic factor only in memory cells would be a novel mechanism of differential gene regulation. This differentiation stage-specific mechanism reflects a basic immunologic principle: naive cells need to expand before becoming an effective defense factor, whereas memory cells with already increased precursor frequency can rapidly mount effector functions to eliminate reinfecting pathogens in a strictly Ag-dependent fashion.

Animals↗

PubMed2006

Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection.

The action of type I IFN (IFN-I) on APCs is well studied, but their direct effect on CD4 T cells is unclear. To address this, we transferred IFN-I receptor-deficient (IFN-IR(0)) and -sufficient (wild-type, WT) TCR-transgenic CD4 T cells into WT mice and analyzed their response to immunization. In response to lymphocytic choriomeningitis virus immunization, WT CD4 T cells expanded approximately 100-fold, whereas IFN-IR(0) CD4 T cells expanded <10-fold. However, both WT and IFN-IR(0) CD4 T cells expanded approximately 10-fold after Listeria monocytogenes immunization. Poor expansion of IFN-IR(0) CD4 T cells after lymphocytic choriomeningitis virus immunization was not due to a defect in proliferation or initial activation but to poor survival of the daughter cells. Thus, direct IFN-I signals can play either a critical or minimal role in CD4 T cell clonal expansion depending on the specific pathogen.

Animals↗

PubMed2005

Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection.

T cell expansion and memory formation are generally more effective when elicited by live organisms than by inactivated vaccines. Elucidation of the underlying mechanisms is important for vaccination and therapeutic strategies. We show that the massive expansion of antigen-specific CD8 T cells that occurs in response to viral infection is critically dependent on the direct action of type I interferons (IFN-Is) on CD8 T cells. By examining the response to infection with lymphocytic choriomeningitis virus using IFN-I receptor-deficient (IFN-IR(0)) and -sufficient CD8 T cells adoptively transferred into normal IFN-IR wild-type hosts, we show that the lack of direct CD8 T cell contact with IFN-I causes >99% reduction in their capacity to expand and generate memory cells. The diminished expansion of IFN-IR(0) CD8 T cells was not caused by a defect in proliferation but by poor survival during the antigen-driven proliferation phase. Thus, IFN-IR signaling in CD8 T cells is critical for the generation of effector and memory cells in response to viral infection.

Animals↗

PubMed2005

Quantifying cell turnover using CFSE data.

The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based "Smith-Martin" model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S+G2+M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett-Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle.

Algorithms↗

PubMed2004

Proliferation and differentiation of CD8+ T cells in the absence of IL-2/15 receptor beta-chain expression or STAT5 activation.

Major gains in the efficacy of T cell-based therapies for cancer and infectious diseases could be realized through improved understanding of the signals that control expansion and differentiation of CD8(+) cytolytic T cells. IL-2, IL-15, and the downstream transcription factor STAT5 have all been implicated as important regulators of these processes, yet there are conflicting data regarding their contribution to in vivo T cell responses. We used a murine adoptive T cell transfer model to examine the contribution of IL-2 and IL-15 signaling to the proliferation and differentiation of naive, CD8(+) T cells bearing an OVA-specific TCR transgene (OT-I). OT-I T cells failed to express the high affinity IL-2R (CD25) while proliferating in vivo, irrespective of the mode of Ag delivery. Moreover, OT-I T cells rendered genetically deficient in the shared IL-2/IL-15Rbeta subunit (IL-2Rbeta) demonstrated normal Ag-induced proliferation and cytolytic activity in vivo. Accordingly, activation of STAT5 was not detected in proliferating IL-2Rbeta-deficient OT-I T cells, thus implicating a STAT5-independent cytokine or costimulatory pathway in this process. Even though IL-2 and IL-15 were dispensable for CD8(+) T cell proliferation, systemic infusion of IL-2 nevertheless promoted the expansion of OT-I T cells in vivo. Thus, IL-2 and IL-15 signals are not essential for CD8(+) T cell proliferation or differentiation, but IL-2 can promote supraphysiological expansion when supplied exogenously. These findings challenge current models that place CD8(+) T cell proliferation under the control of STAT5-dependent cytokines and suggest new approaches to the therapeutic manipulation of T cell numbers in vivo.

Antineoplastic Agents↗

PubMed2004

The Gads (GrpL) adaptor protein regulates T cell homeostasis.

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4(+) and CD8(+) T cells are markedly different. Gads(-/-) CD4(+) T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads(-/-) CD4(+) T cells continued to proliferate at a higher rate than wild-type CD4(+) T cells, demonstrating a defect in homeostatic proliferation. Gads(-/-) CD8(+) T cells had a memory-like phenotype, produced IFN-gamma in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads(-/-) T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads(-/-) CD4(+) T cells, but not CD8(+) T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads(-/-) CD4(+) T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4(+) and CD8(+) T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4(+) T cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The rescaling method for quantifying the turnover of cell populations.

The dynamic nature of immune responses requires the development of appropriate experimental and theoretical tools to quantitatively estimate the division and death rates which determine the turnover of immune cells. A number of papers have used experimental data from BrdU and D-glucose labels together with a simple random birth-death model to quantify the turnover of immune cells focusing on HIV/SIV infections [Mohri et al. 279 (1998) 1223-1227, Hellerstein et al. 5 (1999) 83-89, Bonhoeffer et al. 164 (2000) 5049-5054, Mohri et al. 87 (2001) 1277-1287]. We show how uncertainties in the assumptions of the random birth-death model may lead to substantial errors in the parameters estimated. We then show how more accurate estimates can be obtained from the more recent CFSE data which allow to track the number of divisions each cell has undergone. Specifically, we: (i) describe a general stage-structured model of cell division where the probabilities of division and death are functions of time since the previous division; (ii) develop a rescaling method to identify invariant parameters (i.e. the ones that are independent of the specific functions describing division and death); (iii) show how these invariant parameters can be estimated, and (iv) illustrate this technique by applying it to CFSE data taken from the literature.

Adoptive Transfer↗

PubMed2003

Changing immunodominance patterns in antiviral CD8 T-cell responses after loss of epitope presentation or chronic antigenic stimulation.

The H-2(b)-restricted CD8 T-cell response against lymphocytic choriomeningitis virus is directed against at least 10 dominant and subdominant epitopes, including two newly identified epitopes in the nucleoprotein. We have used this set of epitopes to characterize the plasticity of the hierarchy under different experimental circumstances, i.e., loss of MHC class I molecules, loss of specific epitopes (CTL escape), and prolonged antigenic stimulation (chronic infection). We found that loss of epitope-specific responses was almost inevitably associated with compensatory responses against other, subdominant, epitopes. Multiple epitope loss was required to change the hierarchy. Persistent viral infection was associated with a loss of not only the dominant response against the NP396 epitope, but also a loss of subdominant responses against nucleoprotein epitopes. In contrast, responses against glycoprotein epitopes, dominant and subdominant, survived under chronic infection conditions, and even dominated the response (GP118). Our results suggest that the fate of each specific T-cell response during chronic infection is in part determined by the origin of the cognate epitopes, i.e, the proteins from which they are processed, or, more specifically, nucleoprotein versus glycoprotein. A model in which recruitment time plays a role in the longevity of antiviral T-cell responses during persistent infection is discussed.

Acute Disease↗

PubMed2003

Prolonged presence of effector-memory CD8 T cells in the central nervous system after dengue virus encephalitis.

Dengue virus infection in the central nervous system (CNS) of immunized mice results in a strong influx of CD8 T cells into the brain. Whereas the kinetics of the splenic antiviral response are conventional, i.e. expansion followed by a rapid drop in the frequency of specific CD8 T cells, dengue virus-specific CD8 T cells are retained in the CNS at a high frequency. These CD8 T cells display a partially activated phenotype (CD69(high), Ly-6A/E(high), CD62L(low)), characteristic for effector-memory T cells. CD43 expression, visualized by staining with the 1B11 mAb, decreased in time, suggesting that these persisting CD8 T cells differentiated into memory cells. These data add to the growing evidence implicating the CNS as a non-lymphoid tissue capable of supporting prolonged T cell survival/maintenance.

Animals↗

PubMed2003

Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment.

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.

Animals↗

PubMed2002

Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells.

The generation and efficient maintenance of antigen-specific memory T cells is essential for long-lasting immunological protection. In this study, we examined the role of interleukin (IL)-15 in the generation and maintenance of virus-specific memory CD8 T cells using mice deficient in either IL-15 or the IL-15 receptor alpha chain. Both cytokine- and receptor-deficient mice made potent primary CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV), effectively cleared the virus and generated a pool of antigen-specific memory CD8 T cells that were phenotypically and functionally similar to memory CD8 T cells present in IL-15(+/+) mice. However, longitudinal analysis revealed a slow attrition of virus-specific memory CD8 T cells in the absence of IL-15 signals. This loss of CD8 T cells was due to a severe defect in the proliferative renewal of antigen-specific memory CD8 T cells in IL-15(-/-) mice. Taken together, these results show that IL-15 is not essential for the generation of memory CD8 T cells, but is required for homeostatic proliferation to maintain populations of memory cells over long periods of time.

Animals↗

PubMed2002

Estimating the precursor frequency of naive antigen-specific CD8 T cells.

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.

Adoptive Transfer↗

PubMed2002

Defective generation but normal maintenance of memory T cells in old mice.

The ability to maintain memory after encounter with antigen is one of the central features of the immune system. Immune memory generated during young age can be maintained well into old age. However, we know little about maintenance of immune memory generated during old age. In this study, we compared generation and maintenance of memory CD8 T cells in young (2-3-month-old) and aged (22-24-month-old) mice following acute lymphocytic choriomeningitis virus infection. We found that young mice made a more vigorous primary T cell response and generated higher levels of memory cells than old mice. However, once generated, memory CD8 T cells were maintained at stable levels in both young and old mice for more than 5 months. Interestingly, the generation of a secondary effector response in vivo was again slightly compromised in the old mice. Taken together, these results show that generation of T cell responses is compromised in old age, but maintenance of the pool of memory T cells is not affected by the aging process.

Aging↗