Table_1.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

image_3.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

image_6.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

Table_2.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

image_1.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

image_4.PDF

Background<p>Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.</p>Methods<p>We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.</p>Results<p>M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.</p>Conclusion<p>Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.</p

Image_2_Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses.jpeg

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1β stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1β in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1β driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.</p

Image_1_Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses.jpeg

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1β stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1β in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1β driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.</p

Table_1_Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses.docx

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1β stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1β in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1β driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.</p

Statistical Analysis Plan

Statistical analysis plan for the MTBVAC 201 clinical trial. A phase Ib, randomised, double-blind, dose-escalation clinical trial of the safety, reactogenicity and immunogenicity of MTBVAC compared to BCG Vaccine SSI, in newborns living in a tuberculosis endemic region with a safety arm in adults