Rajshekhar Alli - Academia.edu (original) (raw)
Papers by Rajshekhar Alli
Involvement of NKR-P2/NKG2D in DC-mediated killing of tumor targets: indicative of a common, innate, target-recognition paradigm?
European Journal of Immunology, Apr 1, 2004
DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we de... more DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein‐2 (NKR‐P2) on rat and mouse DC, and we show that NKR‐P2 gets reorganized upon antigen contact. DC activated with anti‐NKR‐P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR‐P2 and its ligand with rNKR‐P2 abrogated apoptotic killing, suggesting NKR‐P2 to function as an activating molecule on DC. In vivo injection of anti‐NKR‐P2 mAb augmented DC activity and delayed tumor progression. NKR‐P2 signaling involved Ca2+ influx, culminating in the expression of the apoptosis‐inducing molecule, TNF‐α. Taken together, these observations suggest that NKR‐P2 (the rat orthologue of human NKG2D) acts as a target‐recognition molecule on DC.
The T cell response to interleukin 10 alters cellular dynamics and paradoxically promotes CNS autoimmunity
The Journal of Immunology, May 1, 2012
FEBS Letters, 2004
Dendritic cells (DCs) are known to modulate immune response by activating e¡ector cells of both t... more Dendritic cells (DCs) are known to modulate immune response by activating e¡ector cells of both the innate and the adaptive immune system. In the present study, we demonstrate that co-culture of DCs with paraformaldehyde-¢xed tumor cells augments the secretion of interleukin (IL)-12 by DCs and these activated DCs upon co-culture with naive NK cells enhance the cytolytic activity of NK cells against NK-sensitive target YAC-1. Similarly, DCs isolated from tumor-bearing animals also activated NK cells in vitro. For e⁄cient activation of NK cells, the ratio of activated DCs to NK cells is crucial. Addition of anti-IL-12 antibody to the culture system completely abolished activation of NK cells by DCs, suggesting that IL-12 secreted by DCs is an essential factor in NK cell activation. Adoptive transfer of DCs isolated from tumor-bearing animals into normal rats also induced activation of NK cells in normal animals.
Indian journal of biochemistry & biophysics, 2002
Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific n... more Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific naïve T cells, B cells and NK cells. Under certain conditions, DCs even silence T cell immune responses in vivo, thus, modulating the immune response. This special function of DCs could be exploited in the treatment of cancer, autoimmune disorder and chronic viral infections.
PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells
Leukemia, Feb 2, 2018
In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeut... more In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.
IL-10 modulates DSS-induced colitis through a macrophage – ROS – NO axis
Breakdown of the epithelial barrier due to toxins or other insults leads to severe colitis. IL-10... more Breakdown of the epithelial barrier due to toxins or other insults leads to severe colitis. IL-10 is a critical regulator of this, yet its cellular targets and mechanisms of action are not resolved. We address this here. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced DSS-induced colitis that did not significantly differ from disease in IL-10−/ − or IL-10Rα−/ − mice; no impact of IL-10Rα-deficiency in other lineages was observed. IL-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Lamina propria macrophages did not show numerical or phenotypic differences from controls, or a competitive advantage over wild type cells. Pro-inflammatory cytokine production, and particularly TNF-α, was increased, though TNF-α neutralization failed to reveal a defining role for this cytokine in the aggravated disease. Rather, IL-10RαMdel lamina propria macrophages produced substantia...
Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity i... more Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3b stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3b loop, stabilizing it. G107 TRBV possess a gap in their CDR3b where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3b stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an,10–1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2 + TCR tested. Analysis of fine specificity indicated a preserved ...
We previously showed that transgenically expressed chimeric Ag-MHC-receptors can Ag-specifically ... more We previously showed that transgenically expressed chimeric Ag-MHC-receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IA s-receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8 ؉ RMTC were therapeutically effective whereas CD4 ؉ RMTC were not. We reexamine here the therapeutic potential of CD4 ؉ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4 ؉ MBP89-101-IA s-RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.
Whereas increased affinity enhances T cell competitiveness after immunization, the role of affini... more Whereas increased affinity enhances T cell competitiveness after immunization, the role of affinity in modulating the pathogenicity of self-reactive T cells is less established. To assess this, we generated two myelin-specific, class II MHC-restricted TCR that differ only in a buried hydroxymethyl that forms a common TCR b-chain V region variant. The variation, predicted to increase TCR stability, resulted in a ∼3log 10 difference in TCR sensitivity with preserved fine specificity. The high-affinity TCR markedly diminished T cell pathogenicity. T cells were not deleted, did not upregulate Foxp3, and barring disease induction were predominantly naive. However, high-affinity CD4 + T cells showed an altered cytokine profile characterized by the production of protective cytokines prior to experimental allergic encephalomyelitis induction and decreased effector cytokines after. Further, the highaffinity TCR promoted the development of CD4 2 CD8 2 and CD8 + T cells that possessed low intrinsic pathogenicity, were protective even in small numbers when transferred into wild-type mice and in mixed chimeras, and outcompete CD4 + T cells during disease development. Therefore, TCR affinities exceeding an upper affinity threshold may impede the development of autoimmunity through altered development and functional maturation of T cells, including diminished intrinsic CD4 + T cell pathogenicity and the development of CD4 2 Foxp3 2 regulatory populations.
The Journal of Immunology
There was an inadvertent error in the labeling of control vector GFP 1 and experimental IL-10R GF... more There was an inadvertent error in the labeling of control vector GFP 1 and experimental IL-10R GFP 1 cohorts in Fig. 8C, and the symbols indicating the different cohorts were reversed. A corrected Fig. 8 with a revised panel (C) is provided below. In addition, the stated p value corresponding to *** in the figure legend was incorrect. The correct value should be ***p # 0.001. The corrected figure legend is also provided below. The authors apologize for any confusion these errors may have caused.
PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells
Leukemia
In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeut... more In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.
European Journal of Immunology, 2004
DC are the most efficient antigen-presenting cells that regulate the immune response. Here, we de... more DC are the most efficient antigen-presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein-2 (NKR-P2) on rat and mouse DC, and we show that NKR-P2 gets reorganized upon antigen contact. DC activated with anti-NKR-P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR-P2 and its ligand with rNKR-P2 abrogated apoptotic killing, suggesting NKR-P2 to function as an activating molecule on DC. In vivo injection of anti-NKR-P2 mAb augmented DC activity and delayed tumor progression. NKR-P2 signaling involved Ca 2+ influx, culminating in the expression of the apoptosis-inducing molecule, TNF- § . Taken together, these observations suggest that NKR-P2 (the rat orthologue of human NKG2D) acts as a target-recognition molecule on DC.
The Journal of Immunology, Apr 1, 2009
We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin ... more We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoproteinspecific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag ؊/؊ mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application. Abbreviations used in this paper: pMHC, peptide MHC; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; rhIL-2, recombinant human IL-2.
Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation
Journal of immunology (Baltimore, Md. : 1950), Jan 24, 2016
How a large number of cytokines differentially signal through a small number of signal transducti... more How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th1...
Dendritic cells as natural adjuvants and modulators of immune response in cancer immunotherapy
Indian journal of biochemistry & biophysics, 2002
Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific n... more Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific naïve T cells, B cells and NK cells. Under certain conditions, DCs even silence T cell immune responses in vivo, thus, modulating the immune response. This special function of DCs could be exploited in the treatment of cancer, autoimmune disorder and chronic viral infections.
IL-10-dependent infectious tolerance after the treatment of experimental allergic encephalomyelitis with redirected CD4+CD25+ T lymphocytes
Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2005
How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclea... more How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclear. In this report we analyze the immunomodulatory activity of CD4+CD25+ T cells that are antigen-specifically redirected against myelin basic protein (MBP)89-101-specific autoreactive T cells by a MBP89-101-IA(s)-zeta chimeric receptor. We have previously shown that these redirected regulatory T cells are highly potent in treating a model autoimmune disease, experimental allergic encephalomyelitis. We show here that they have only limited effect in vivo on autoreactive T cell proliferation and therefore do not act by deleting or suppressing the expansion of pathologic effector cells. Rather, the redirected CD4+CD25+ T cells divert the pathologic T helper 1 self-specific T cell response to one characterized by high IL-10 and lower IL-4 production. Significantly, when isolated from the inducing CD4+CD25+ regulatory T cells, these self-specific T cells can independently suppress the autorea...
Indian Journal of Clinical Biochemistry, 1999
Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other dev... more Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other developing tropical countries. One of the major lacunae in the effective management of clinical filarial cases is the non-availability of a suitable diagnostic test for confirming filaria aetiology in acute, chronic and occult clinical cases where microfilariae (mf) are not usually seen in peripheral circulation. Studies in our laboratory have shown the usefulness of filarial antibody and antigen assays using microfilarial excretory-secretory (mr ES) antigen in detecting microfilaraemic, acute and chronic filarial cases and in confirming filarial aetiology in occult infections. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been explored in the treatment of microfilaraemic cases. Immunomonitoring has shown that the seroconversion of antigen and antibody positivity was found to be very helpful in determining appropriate period of DEC treatment for clinical relief and cure in clinical filarial patients and further they did not have recurrence in most of the cases. Optimal DEC (6mg/kg body wtlday for 21 days each month for 3-12 months) therapy was found to be very effective in acute and atypical clinical manifestations such as asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent upper respiratory tract infections (URI), pneumonia (super imposed infections) in children and minimal hydrocele, epididymoorchitis, lymphangitis, lymphadenitis, acute abdomen, central serous retinopathy, tenosynovitis, pain and swelling in limbs and joints in adults living in filaria endemic areas. KEY WORDS : Filariasis, immunodiagnosis, immunomonitoring, DEC therapy. FILARIASIS -A MAJOR PUBLIC HEALTH PROBLEM Human lymphatic filariasis is caused by the nematode parasites Wuchereria bancrofti and Brugia malayi. Filariasis is a mosquito borne disease with infected man serving as a reservoir.
Indian Journal of Clinical Biochemistry, 1999
Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained by in vitro maintenance... more Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained by in vitro maintenance of mf are important tools in the immunodiagnosis of bancroftian filariasis.
Indian Journal of Clinical Biochemistry, 2003
Indian Journal of Clinical Biochemistry, 2001
A comparative analysis was made on the utility of SEVAFILACHEK-stick based immunoassays and comme... more A comparative analysis was made on the utility of SEVAFILACHEK-stick based immunoassays and commercially available ICT-filariasis test to detect active infection in different groups of bancroftian filadasis. The SEVAFILACHEK immunoassays were found to be useful to detect filarial infection in microfilaraemia and in a significant number of clinical filarial cases with acute, chronic and occult clinical manifestations. In the clinical cases, microfilariae are not usually detected in peripheral circulation. Employing SEVAFILACHEK assays 6 and 6 of the7 samples of patients with chronic filarial disease, and s and 6 of tS microfilaraemic cases gave positivity for fllarial IgG antibodies and antigen respectively. Four of the IS occult filarial samples were positive for antibodies and antigen. Filarial antigen was detected by ICT-filariasis test in blood samples of all the IS microfilariaemic cases, 1 chronic filarial and 2 occult filarial samples. The main advantage of ICT assay is its rapid format and convenience for field use.
Involvement of NKR-P2/NKG2D in DC-mediated killing of tumor targets: indicative of a common, innate, target-recognition paradigm?
European Journal of Immunology, Apr 1, 2004
DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we de... more DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein‐2 (NKR‐P2) on rat and mouse DC, and we show that NKR‐P2 gets reorganized upon antigen contact. DC activated with anti‐NKR‐P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR‐P2 and its ligand with rNKR‐P2 abrogated apoptotic killing, suggesting NKR‐P2 to function as an activating molecule on DC. In vivo injection of anti‐NKR‐P2 mAb augmented DC activity and delayed tumor progression. NKR‐P2 signaling involved Ca2+ influx, culminating in the expression of the apoptosis‐inducing molecule, TNF‐α. Taken together, these observations suggest that NKR‐P2 (the rat orthologue of human NKG2D) acts as a target‐recognition molecule on DC.
The T cell response to interleukin 10 alters cellular dynamics and paradoxically promotes CNS autoimmunity
The Journal of Immunology, May 1, 2012
FEBS Letters, 2004
Dendritic cells (DCs) are known to modulate immune response by activating e¡ector cells of both t... more Dendritic cells (DCs) are known to modulate immune response by activating e¡ector cells of both the innate and the adaptive immune system. In the present study, we demonstrate that co-culture of DCs with paraformaldehyde-¢xed tumor cells augments the secretion of interleukin (IL)-12 by DCs and these activated DCs upon co-culture with naive NK cells enhance the cytolytic activity of NK cells against NK-sensitive target YAC-1. Similarly, DCs isolated from tumor-bearing animals also activated NK cells in vitro. For e⁄cient activation of NK cells, the ratio of activated DCs to NK cells is crucial. Addition of anti-IL-12 antibody to the culture system completely abolished activation of NK cells by DCs, suggesting that IL-12 secreted by DCs is an essential factor in NK cell activation. Adoptive transfer of DCs isolated from tumor-bearing animals into normal rats also induced activation of NK cells in normal animals.
Indian journal of biochemistry & biophysics, 2002
Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific n... more Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific naïve T cells, B cells and NK cells. Under certain conditions, DCs even silence T cell immune responses in vivo, thus, modulating the immune response. This special function of DCs could be exploited in the treatment of cancer, autoimmune disorder and chronic viral infections.
PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells
Leukemia, Feb 2, 2018
In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeut... more In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.
IL-10 modulates DSS-induced colitis through a macrophage – ROS – NO axis
Breakdown of the epithelial barrier due to toxins or other insults leads to severe colitis. IL-10... more Breakdown of the epithelial barrier due to toxins or other insults leads to severe colitis. IL-10 is a critical regulator of this, yet its cellular targets and mechanisms of action are not resolved. We address this here. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced DSS-induced colitis that did not significantly differ from disease in IL-10−/ − or IL-10Rα−/ − mice; no impact of IL-10Rα-deficiency in other lineages was observed. IL-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Lamina propria macrophages did not show numerical or phenotypic differences from controls, or a competitive advantage over wild type cells. Pro-inflammatory cytokine production, and particularly TNF-α, was increased, though TNF-α neutralization failed to reveal a defining role for this cytokine in the aggravated disease. Rather, IL-10RαMdel lamina propria macrophages produced substantia...
Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity i... more Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3b stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3b loop, stabilizing it. G107 TRBV possess a gap in their CDR3b where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3b stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an,10–1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2 + TCR tested. Analysis of fine specificity indicated a preserved ...
We previously showed that transgenically expressed chimeric Ag-MHC-receptors can Ag-specifically ... more We previously showed that transgenically expressed chimeric Ag-MHC-receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IA s-receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8 ؉ RMTC were therapeutically effective whereas CD4 ؉ RMTC were not. We reexamine here the therapeutic potential of CD4 ؉ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4 ؉ MBP89-101-IA s-RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.
Whereas increased affinity enhances T cell competitiveness after immunization, the role of affini... more Whereas increased affinity enhances T cell competitiveness after immunization, the role of affinity in modulating the pathogenicity of self-reactive T cells is less established. To assess this, we generated two myelin-specific, class II MHC-restricted TCR that differ only in a buried hydroxymethyl that forms a common TCR b-chain V region variant. The variation, predicted to increase TCR stability, resulted in a ∼3log 10 difference in TCR sensitivity with preserved fine specificity. The high-affinity TCR markedly diminished T cell pathogenicity. T cells were not deleted, did not upregulate Foxp3, and barring disease induction were predominantly naive. However, high-affinity CD4 + T cells showed an altered cytokine profile characterized by the production of protective cytokines prior to experimental allergic encephalomyelitis induction and decreased effector cytokines after. Further, the highaffinity TCR promoted the development of CD4 2 CD8 2 and CD8 + T cells that possessed low intrinsic pathogenicity, were protective even in small numbers when transferred into wild-type mice and in mixed chimeras, and outcompete CD4 + T cells during disease development. Therefore, TCR affinities exceeding an upper affinity threshold may impede the development of autoimmunity through altered development and functional maturation of T cells, including diminished intrinsic CD4 + T cell pathogenicity and the development of CD4 2 Foxp3 2 regulatory populations.
The Journal of Immunology
There was an inadvertent error in the labeling of control vector GFP 1 and experimental IL-10R GF... more There was an inadvertent error in the labeling of control vector GFP 1 and experimental IL-10R GFP 1 cohorts in Fig. 8C, and the symbols indicating the different cohorts were reversed. A corrected Fig. 8 with a revised panel (C) is provided below. In addition, the stated p value corresponding to *** in the figure legend was incorrect. The correct value should be ***p # 0.001. The corrected figure legend is also provided below. The authors apologize for any confusion these errors may have caused.
PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells
Leukemia
In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeut... more In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.
European Journal of Immunology, 2004
DC are the most efficient antigen-presenting cells that regulate the immune response. Here, we de... more DC are the most efficient antigen-presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein-2 (NKR-P2) on rat and mouse DC, and we show that NKR-P2 gets reorganized upon antigen contact. DC activated with anti-NKR-P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR-P2 and its ligand with rNKR-P2 abrogated apoptotic killing, suggesting NKR-P2 to function as an activating molecule on DC. In vivo injection of anti-NKR-P2 mAb augmented DC activity and delayed tumor progression. NKR-P2 signaling involved Ca 2+ influx, culminating in the expression of the apoptosis-inducing molecule, TNF- § . Taken together, these observations suggest that NKR-P2 (the rat orthologue of human NKG2D) acts as a target-recognition molecule on DC.
The Journal of Immunology, Apr 1, 2009
We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin ... more We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoproteinspecific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag ؊/؊ mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application. Abbreviations used in this paper: pMHC, peptide MHC; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; rhIL-2, recombinant human IL-2.
Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation
Journal of immunology (Baltimore, Md. : 1950), Jan 24, 2016
How a large number of cytokines differentially signal through a small number of signal transducti... more How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th1...
Dendritic cells as natural adjuvants and modulators of immune response in cancer immunotherapy
Indian journal of biochemistry & biophysics, 2002
Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific n... more Dendritic cells (DCs) are potent antigen presenting cells. Mature DCs activate antigen specific naïve T cells, B cells and NK cells. Under certain conditions, DCs even silence T cell immune responses in vivo, thus, modulating the immune response. This special function of DCs could be exploited in the treatment of cancer, autoimmune disorder and chronic viral infections.
IL-10-dependent infectious tolerance after the treatment of experimental allergic encephalomyelitis with redirected CD4+CD25+ T lymphocytes
Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2005
How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclea... more How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclear. In this report we analyze the immunomodulatory activity of CD4+CD25+ T cells that are antigen-specifically redirected against myelin basic protein (MBP)89-101-specific autoreactive T cells by a MBP89-101-IA(s)-zeta chimeric receptor. We have previously shown that these redirected regulatory T cells are highly potent in treating a model autoimmune disease, experimental allergic encephalomyelitis. We show here that they have only limited effect in vivo on autoreactive T cell proliferation and therefore do not act by deleting or suppressing the expansion of pathologic effector cells. Rather, the redirected CD4+CD25+ T cells divert the pathologic T helper 1 self-specific T cell response to one characterized by high IL-10 and lower IL-4 production. Significantly, when isolated from the inducing CD4+CD25+ regulatory T cells, these self-specific T cells can independently suppress the autorea...
Indian Journal of Clinical Biochemistry, 1999
Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other dev... more Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other developing tropical countries. One of the major lacunae in the effective management of clinical filarial cases is the non-availability of a suitable diagnostic test for confirming filaria aetiology in acute, chronic and occult clinical cases where microfilariae (mf) are not usually seen in peripheral circulation. Studies in our laboratory have shown the usefulness of filarial antibody and antigen assays using microfilarial excretory-secretory (mr ES) antigen in detecting microfilaraemic, acute and chronic filarial cases and in confirming filarial aetiology in occult infections. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been explored in the treatment of microfilaraemic cases. Immunomonitoring has shown that the seroconversion of antigen and antibody positivity was found to be very helpful in determining appropriate period of DEC treatment for clinical relief and cure in clinical filarial patients and further they did not have recurrence in most of the cases. Optimal DEC (6mg/kg body wtlday for 21 days each month for 3-12 months) therapy was found to be very effective in acute and atypical clinical manifestations such as asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent upper respiratory tract infections (URI), pneumonia (super imposed infections) in children and minimal hydrocele, epididymoorchitis, lymphangitis, lymphadenitis, acute abdomen, central serous retinopathy, tenosynovitis, pain and swelling in limbs and joints in adults living in filaria endemic areas. KEY WORDS : Filariasis, immunodiagnosis, immunomonitoring, DEC therapy. FILARIASIS -A MAJOR PUBLIC HEALTH PROBLEM Human lymphatic filariasis is caused by the nematode parasites Wuchereria bancrofti and Brugia malayi. Filariasis is a mosquito borne disease with infected man serving as a reservoir.
Indian Journal of Clinical Biochemistry, 1999
Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained by in vitro maintenance... more Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained by in vitro maintenance of mf are important tools in the immunodiagnosis of bancroftian filariasis.
Indian Journal of Clinical Biochemistry, 2003
Indian Journal of Clinical Biochemistry, 2001
A comparative analysis was made on the utility of SEVAFILACHEK-stick based immunoassays and comme... more A comparative analysis was made on the utility of SEVAFILACHEK-stick based immunoassays and commercially available ICT-filariasis test to detect active infection in different groups of bancroftian filadasis. The SEVAFILACHEK immunoassays were found to be useful to detect filarial infection in microfilaraemia and in a significant number of clinical filarial cases with acute, chronic and occult clinical manifestations. In the clinical cases, microfilariae are not usually detected in peripheral circulation. Employing SEVAFILACHEK assays 6 and 6 of the7 samples of patients with chronic filarial disease, and s and 6 of tS microfilaraemic cases gave positivity for fllarial IgG antibodies and antigen respectively. Four of the IS occult filarial samples were positive for antibodies and antigen. Filarial antigen was detected by ICT-filariasis test in blood samples of all the IS microfilariaemic cases, 1 chronic filarial and 2 occult filarial samples. The main advantage of ICT assay is its rapid format and convenience for field use.