Objective-Identify the etiology of elevated B 12 in autoimmune lymphoproliferative syndrome (ALPS... more Objective-Identify the etiology of elevated B 12 in autoimmune lymphoproliferative syndrome (ALPS). Design-Peripheral blood of ALPS patients with elevated B 12 and controls were evaluated. Results-Total and holo-haptocorrin (HC) levels were 26-and 23-fold higher in ALPS patients, respectively. No abnormal B 12-binding proteins were found. Western blot revealed HC in lymphocyte lysates only from ALPS patients. Conclusion-Elevated concentrations of B 12 found in ALPS patients were due to increased lymphocyte expression of HC.
Elimination of autoreactive CD4 þ T cells through the death receptor Fas/CD95 is an important mec... more Elimination of autoreactive CD4 þ T cells through the death receptor Fas/CD95 is an important mechanism of immunological selftolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4 þ T cells with an effector memory phenotype (effector memory T cells (T EM)), whereas central memory and activated naïve CD4 þ T cells are relatively resistant to both. Sensitivity of T EM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate T EM linked to the pathogenesis of a number of autoimmune diseases.
To describe inflammatory ocular findings in patients with autoimmune lymphoproliferative syndrome... more To describe inflammatory ocular findings in patients with autoimmune lymphoproliferative syndrome (ALPS). Methods: Retrospective review of medical records for ALPS patients seen at National Eye Institute between 2003 and 2013. Results: Twenty-nine ALPS patients previously referred for ocular or visual symptoms or history of prolonged corticosteroid use were identified. Mean age was 20 years (range, 4-66 years). The majority were male (n = 21, 72.4%) and Caucasian (n = 24, 82.8%). Ten (34.5%) had abnormal ocular findings, the most common of which was an ocular inflammatory disorder (n = 4, 13.8%). Uveitis was seen in two patients with ALPS-FAS and one with ALPS-U, all of whom required long-term systemic immunosuppression. One patient with ALPS-FAS had history of optic neuritis. Conclusions: ALPS can have intraocular inflammatory manifestations that require routine follow-up to ensure appropriate and timely treatment of intraocular disease. Long-term immunosuppression may be needed for patients with ALPS-associated uveitis.
Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in... more Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.
CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimul... more CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling via elevated levels of JAK2 and LYN.
Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of apoptosis. It is frequently ... more Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of apoptosis. It is frequently caused by mutations in FAS (TNFRSF6) gene. Unlike most of the selflimiting autoimmune cytopenias sporadically seen in childhood, multi lineage cytopenias due to ALPS are often refractory, as their inherited genetic defect is not going to go away. Historically, more ALPS patients have died due to overwhelming sepsis following splenectomy to manage their chronic cytopenias than due to any other cause, including malignancies. Hence, current recommendations underscore the importance of avoiding splenectomy in ALPS, by long-term use of corticosteroid-sparing immunosuppressive agents like mycophenolate mofetil and sirolimus. Paradigms learnt from managing ALPS patients in recent years is highlighted here and can be extrapolated to manage refractory cytopenias in patients with as yet undetermined genetic bases for their ailments. It is also desirable to develop international registries for children with rare and complex immune problems associated with chronic multilineage cytopenias in order to elucidate their natural history and long-term comorbidities due to the disease and its treatments.
METHODS. We performed an observational natural history study of 50 patients with CTLA4h who were ... more METHODS. We performed an observational natural history study of 50 patients with CTLA4h who were followed at the NIH. We analyzed clinical, radiological, immunological, and histopathological data. RESULTS. Evidence for neuroinflammation was observed in 32% (n = 16 of 50) of patients in this cohort by magnetic resonance imaging (MRI) and/or by cerebrospinal fluid analysis. Clinical symptoms were commonly absent or mild in severity, with headaches as the leading complaint (n = 13 of 16). The most striking findings were relapsing, large, contrastenhancing focal lesions in the brain and spinal cord observed on MRI. We detected inflammation in the cerebrospinal fluid and leptomeninges before the parenchyma. Brain biopsies of inflammatory lesions from 10 patients showed perivascular and intraparenchymal mixed cellular infiltrates with little accompanying demyelination or neuronal injury. CONCLUSIONS. Neuroinflammation due to CTLA4h is mediated primarily by an infiltrative process with a distinct and striking dissociation between clinical symptoms and radiological findings in the majority of patients.
Background: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-f... more Background: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. Objective: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. Methods: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. Results: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD42CD82) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. Conclusion: : STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome. (J Allergy Clin Immunol 2022;nnn:nnn-nnn.)
Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of com... more Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of common variable immunodeficiency (CVID). However, the causal link between NFKB1 variants and CVID has not been demonstrated experimentally and genetically, and there has been insufficient biochemical characterization and enrichment analysis. We show that the cotransfection of NFKB1-deficient HEK293T cells (lacking both p105 and its cleaved form p50) with a κB reporter, NFKB1/p105, and a homodimerization-defective RELA/p65 mutant results in p50:p65 heterodimer–dependent and p65:p65 homodimer–independent transcriptional activation. We found that 59 of the 90 variants in patients with CVID or related conditions were loss of function or hypomorphic. By contrast, 258 of 260 variants in the general population or patients with unrelated conditions were neutral. None of the deleterious variants displayed negative dominance. The enrichment in deleterious NFKB1 variants of patients with CVID was sele...
Haematopoietic stem cell transplant (HSCT) is the treatment of choice in young patients (age < 50... more Haematopoietic stem cell transplant (HSCT) is the treatment of choice in young patients (age < 50 years) with aplastic anaemia [AA]. We analysed the influence of telomere length (TL) in modulating the outcome of AA patients undegoing HSCT. Methods: Patients undergoing HSCT for AA at CMC Vellore between 2004 and 2017 where DNA samples were available were included in this study. Clinical parameters were documented. DNA was extracted from recipient (pre-HSCT) and their donors and telomere length was measured using quantitative real-time PCR (qPCR) using relative quantitation method. The association between telomere length (TL) and clinical parameters were analysed using SPSS Software. Results: DNA was available in 170 out of 252 patients. 137 had sibling/family donors while 9 had MUD and 24 haploidentical donors. Main graft source was PBSC (95%) and 58% received fludarabine based conditioning. The incidence of graft failure, aGVHD and chGVHD were 13% (n = 22), 25% (n = 43) and 23% (n = 39) respectively. Patients had significantly lower relative TL as compared to donors [Median pTL-.79 (range: .07-4.08) versus dTL-.87 (range: .04-5.76) P = .032]. TL was categorized into long (>.87) or short (<.87) based on the median TL. Median time to engraftment was shorter in donors with higher TL (14 versus 15 days P = .031) but was not associated with aGVHD. IN MRD donors, incidence of graft failure was 11.2% and was significantly higher when donor had short TL [19.2% versus 4.5%; P = .018]. Logistic regression analysis showed long donor TL to be associated with faster engraftment (P = .041) and better outcomes (P = .031; multivariate: P = .035). Conclusion: Donor telomere length may play an important role in engraftment, graft failure and overall survival in patients undergoing matched related donor transplant for aplastic anaemia. Newer strategies may need to be considered.
Elucidating the molecular basis of immunodeficiency diseases is a powerful approach to discoverin... more Elucidating the molecular basis of immunodeficiency diseases is a powerful approach to discovering new immunoregulatory pathways in humans. Here we report 10 affected individuals from 4 families with a new immunodeficiency disease comprising of severe progressive lymphopenia, autoimmunity, immunodeficiency, and liver disease due to recessive loss of function variants in “GTPase of immunity-associated proteins” protein 5 (GIMAP5). We show that the disease involves the progressive loss of naïve T lymphocytes and a corresponding increase in antigen-experienced, but poorly functional and replicatively senescent T cells. In vivo treatment of Gimap5-deficient mice with rapamycin (an inhibitor of mTORC1) significantly restores the fraction of naïve T lymphocytes. Furthermore, a GIMAP5-deficient human patient who was treated with rapamycin (sirolimus) showed a remarkable reduction in spleen/lymph node size. Together, these observations reveal that GIMAP5 plays a critical role in lymphocyte ...
We describe the integration of multi-batch cytometry datasets (iMUBAC), a flexible, robust, and s... more We describe the integration of multi-batch cytometry datasets (iMUBAC), a flexible, robust, and scalable computational framework for unsupervised cell-type identification across multiple batches of high-dimensional cytometry datasets. After overlaying cells from healthy controls across multiple batches, iMUBAC learns batch-specific cell-type classification boundaries and identifies aberrant immunophenotypes in patient samples. We illustrate unbiased and streamlined immunophenotyping, using both in-house and public mass and flow cytometry datasets.
Objective-Identify the etiology of elevated B 12 in autoimmune lymphoproliferative syndrome (ALPS... more Objective-Identify the etiology of elevated B 12 in autoimmune lymphoproliferative syndrome (ALPS). Design-Peripheral blood of ALPS patients with elevated B 12 and controls were evaluated. Results-Total and holo-haptocorrin (HC) levels were 26-and 23-fold higher in ALPS patients, respectively. No abnormal B 12-binding proteins were found. Western blot revealed HC in lymphocyte lysates only from ALPS patients. Conclusion-Elevated concentrations of B 12 found in ALPS patients were due to increased lymphocyte expression of HC.
Elimination of autoreactive CD4 þ T cells through the death receptor Fas/CD95 is an important mec... more Elimination of autoreactive CD4 þ T cells through the death receptor Fas/CD95 is an important mechanism of immunological selftolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4 þ T cells with an effector memory phenotype (effector memory T cells (T EM)), whereas central memory and activated naïve CD4 þ T cells are relatively resistant to both. Sensitivity of T EM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate T EM linked to the pathogenesis of a number of autoimmune diseases.
To describe inflammatory ocular findings in patients with autoimmune lymphoproliferative syndrome... more To describe inflammatory ocular findings in patients with autoimmune lymphoproliferative syndrome (ALPS). Methods: Retrospective review of medical records for ALPS patients seen at National Eye Institute between 2003 and 2013. Results: Twenty-nine ALPS patients previously referred for ocular or visual symptoms or history of prolonged corticosteroid use were identified. Mean age was 20 years (range, 4-66 years). The majority were male (n = 21, 72.4%) and Caucasian (n = 24, 82.8%). Ten (34.5%) had abnormal ocular findings, the most common of which was an ocular inflammatory disorder (n = 4, 13.8%). Uveitis was seen in two patients with ALPS-FAS and one with ALPS-U, all of whom required long-term systemic immunosuppression. One patient with ALPS-FAS had history of optic neuritis. Conclusions: ALPS can have intraocular inflammatory manifestations that require routine follow-up to ensure appropriate and timely treatment of intraocular disease. Long-term immunosuppression may be needed for patients with ALPS-associated uveitis.
Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in... more Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.
CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimul... more CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling via elevated levels of JAK2 and LYN.
Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of apoptosis. It is frequently ... more Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of apoptosis. It is frequently caused by mutations in FAS (TNFRSF6) gene. Unlike most of the selflimiting autoimmune cytopenias sporadically seen in childhood, multi lineage cytopenias due to ALPS are often refractory, as their inherited genetic defect is not going to go away. Historically, more ALPS patients have died due to overwhelming sepsis following splenectomy to manage their chronic cytopenias than due to any other cause, including malignancies. Hence, current recommendations underscore the importance of avoiding splenectomy in ALPS, by long-term use of corticosteroid-sparing immunosuppressive agents like mycophenolate mofetil and sirolimus. Paradigms learnt from managing ALPS patients in recent years is highlighted here and can be extrapolated to manage refractory cytopenias in patients with as yet undetermined genetic bases for their ailments. It is also desirable to develop international registries for children with rare and complex immune problems associated with chronic multilineage cytopenias in order to elucidate their natural history and long-term comorbidities due to the disease and its treatments.
METHODS. We performed an observational natural history study of 50 patients with CTLA4h who were ... more METHODS. We performed an observational natural history study of 50 patients with CTLA4h who were followed at the NIH. We analyzed clinical, radiological, immunological, and histopathological data. RESULTS. Evidence for neuroinflammation was observed in 32% (n = 16 of 50) of patients in this cohort by magnetic resonance imaging (MRI) and/or by cerebrospinal fluid analysis. Clinical symptoms were commonly absent or mild in severity, with headaches as the leading complaint (n = 13 of 16). The most striking findings were relapsing, large, contrastenhancing focal lesions in the brain and spinal cord observed on MRI. We detected inflammation in the cerebrospinal fluid and leptomeninges before the parenchyma. Brain biopsies of inflammatory lesions from 10 patients showed perivascular and intraparenchymal mixed cellular infiltrates with little accompanying demyelination or neuronal injury. CONCLUSIONS. Neuroinflammation due to CTLA4h is mediated primarily by an infiltrative process with a distinct and striking dissociation between clinical symptoms and radiological findings in the majority of patients.
Background: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-f... more Background: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. Objective: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. Methods: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. Results: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD42CD82) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. Conclusion: : STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome. (J Allergy Clin Immunol 2022;nnn:nnn-nnn.)
Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of com... more Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of common variable immunodeficiency (CVID). However, the causal link between NFKB1 variants and CVID has not been demonstrated experimentally and genetically, and there has been insufficient biochemical characterization and enrichment analysis. We show that the cotransfection of NFKB1-deficient HEK293T cells (lacking both p105 and its cleaved form p50) with a κB reporter, NFKB1/p105, and a homodimerization-defective RELA/p65 mutant results in p50:p65 heterodimer–dependent and p65:p65 homodimer–independent transcriptional activation. We found that 59 of the 90 variants in patients with CVID or related conditions were loss of function or hypomorphic. By contrast, 258 of 260 variants in the general population or patients with unrelated conditions were neutral. None of the deleterious variants displayed negative dominance. The enrichment in deleterious NFKB1 variants of patients with CVID was sele...
Haematopoietic stem cell transplant (HSCT) is the treatment of choice in young patients (age < 50... more Haematopoietic stem cell transplant (HSCT) is the treatment of choice in young patients (age < 50 years) with aplastic anaemia [AA]. We analysed the influence of telomere length (TL) in modulating the outcome of AA patients undegoing HSCT. Methods: Patients undergoing HSCT for AA at CMC Vellore between 2004 and 2017 where DNA samples were available were included in this study. Clinical parameters were documented. DNA was extracted from recipient (pre-HSCT) and their donors and telomere length was measured using quantitative real-time PCR (qPCR) using relative quantitation method. The association between telomere length (TL) and clinical parameters were analysed using SPSS Software. Results: DNA was available in 170 out of 252 patients. 137 had sibling/family donors while 9 had MUD and 24 haploidentical donors. Main graft source was PBSC (95%) and 58% received fludarabine based conditioning. The incidence of graft failure, aGVHD and chGVHD were 13% (n = 22), 25% (n = 43) and 23% (n = 39) respectively. Patients had significantly lower relative TL as compared to donors [Median pTL-.79 (range: .07-4.08) versus dTL-.87 (range: .04-5.76) P = .032]. TL was categorized into long (>.87) or short (<.87) based on the median TL. Median time to engraftment was shorter in donors with higher TL (14 versus 15 days P = .031) but was not associated with aGVHD. IN MRD donors, incidence of graft failure was 11.2% and was significantly higher when donor had short TL [19.2% versus 4.5%; P = .018]. Logistic regression analysis showed long donor TL to be associated with faster engraftment (P = .041) and better outcomes (P = .031; multivariate: P = .035). Conclusion: Donor telomere length may play an important role in engraftment, graft failure and overall survival in patients undergoing matched related donor transplant for aplastic anaemia. Newer strategies may need to be considered.
Elucidating the molecular basis of immunodeficiency diseases is a powerful approach to discoverin... more Elucidating the molecular basis of immunodeficiency diseases is a powerful approach to discovering new immunoregulatory pathways in humans. Here we report 10 affected individuals from 4 families with a new immunodeficiency disease comprising of severe progressive lymphopenia, autoimmunity, immunodeficiency, and liver disease due to recessive loss of function variants in “GTPase of immunity-associated proteins” protein 5 (GIMAP5). We show that the disease involves the progressive loss of naïve T lymphocytes and a corresponding increase in antigen-experienced, but poorly functional and replicatively senescent T cells. In vivo treatment of Gimap5-deficient mice with rapamycin (an inhibitor of mTORC1) significantly restores the fraction of naïve T lymphocytes. Furthermore, a GIMAP5-deficient human patient who was treated with rapamycin (sirolimus) showed a remarkable reduction in spleen/lymph node size. Together, these observations reveal that GIMAP5 plays a critical role in lymphocyte ...
We describe the integration of multi-batch cytometry datasets (iMUBAC), a flexible, robust, and s... more We describe the integration of multi-batch cytometry datasets (iMUBAC), a flexible, robust, and scalable computational framework for unsupervised cell-type identification across multiple batches of high-dimensional cytometry datasets. After overlaying cells from healthy controls across multiple batches, iMUBAC learns batch-specific cell-type classification boundaries and identifies aberrant immunophenotypes in patient samples. We illustrate unbiased and streamlined immunophenotyping, using both in-house and public mass and flow cytometry datasets.
Uploads
Papers by Koneti Rao