IMMUNOLOGICAL MEMORY CD4 T Follicular Helper Cells Memory CD8 T Cell Differentiation
CD4 T Cell Differentiation Bcl-6 T-bet GATA-3 ROR t Foxp3
CD4 T follicular helper (Tfh) cells FUNCTION Provide essential help for germinal center formation and maintenance Required for affinity maturation and differentiation of memory B cells and long-lived plasma cells PHENOTYPE The chemokine receptor CXCR5 is a functional marker for CD4+ Tfh cells Bcl6 is a lineage regulator of Tfh differentiation in CD4 T cells PD-1, ICOS other phenotypic markers Cytokines IL-21, (IL-4)
Role of CD4 T cell help in antibody responses
CD4 T cells are required for maintenance of the GC reaction CD4 T cells were depleted when GC were established (Day 15 post-lcmv infection)
Ruins of GC remain after CD4 depletion CD4 T cells were depleted when GC were established (Day 15 post-lcmv infection). Mice were sacrificed 7 days after CD4 T cell depletion.
IL-21 is needed for long-term humoral immunity after acute viral infection LCMV specific IgG (log 2) WT IL-21R KO WT IL-21R KO Days post-infection Mohammed et al. J Virol, 2013 Similar results seen with influenza virus and VSV
Importance of IL-21 for generation of long-lived plasma cells Spleen ASC in spleen (day 300) WT KO Day 8 Day 15 Day 22 Day 38 Day 300 Bone Marrow ASC in BM (day 300) Day 8 Day 15 Day 22 Day 38 Day 300 WT KO
Is there a Tfh memory cell? Central Questions: 1. Are there memory CD4 T cells that are poised to recall Tfh lineagespecific functions upon reencounter with antigen? 2. What are markers that define Tfh memory cells? 3. What is the tissue distribution of Tfh effector and Tfh memory cells? 1. What are mechanisms that reinforce the lineage-specific functions of memory Tfh cells? These questions are important for rational vaccine design, where improving the generation and engagement of memory Tfh cells could enhance vaccine-induced immunity. Hale et al. Immunity 2013
Do CXCR5+ memory cells preferentially recall Tfh effector responses following antigen reencounter?
CXCR5+ memory cells recall Tfh secondary effector cells
CXCR5+ memory cells promote enhanced germinal center B cell responses following viral challenge Day 7 Gated on CD19+B220+ Splenocytes
Distinct Tfh and Th1 memory CD4 T cell subsets exist that recall their lineage-specific functions upon reencounter with antigen
Memory CD8 T cell differentiation Effector Memory Naive
CD8 T cells migrate to many nonlymphoid tissues while effectors Lymphoid tissue Naive Effector Memory Intestinal mucosa a4b7 CCR9 And become resident
Resident memory T cells Observed in many tissues Small intestine epithelium Skin Lung Salivary gland Do not recirculate with other compartments Are not found in blood
Intestinal mucosa Lymphoid tissue Environmental milieu regulates phenotype and maintenance of resident memory CD8 T cells Naive Effector CD62L lo Memory CD62L hi Memory CD62L dependent Re-circulation LN Casey, et al., J Immunol 2012 TGF IL-33? TNFa? CD103 7 Ly6C GrB CD69 Survival CD103/ 7 dependent residence
Function of resident memory CD8 T cells extend beyond local killing of infected cells
Sensing and alarm function: when resident memory CD8 T cells encounter Ag in tissues: Blood Cognate antigen Res Memory T cell CD 8 CD 8 Virus or peptide Schenkel, et al., Nat Immun 2013 CD 8
Sensing and alarm function: when resident memory CD8 T cells encounter Ag in tissues: they rapidly induce local chemokine expression Blood Cognate antigen Res Memory T cell CD 8 IFN Chemokines CD 8 Virus or peptide Schenkel, et al., Nat Immun 2013 CD 8
Sensing and alarm function: when resident memory CD8 T cells encounter Ag in tissues: they rapidly recruit additional memory CD8 T cells Blood Cognate antigen Res Memory T cell CD 8 CD 8 CD 8 IFN CD 8 Chemokines CD 8 CD 8 CD 8 Virus or peptide CD 8 CD 8 Schenkel, et al., Nat Immun 2013 CD 8
Protective Immunity by Memory CD8 T cells: Division of Labor Resident memory CD8 T cells at mucosal sites immediate effector function limited proliferative capacity Circulating memory CD8 T cells in blood and lymphoid tissue can differentiate into effectors rapidly substantial proliferative capacity
Yellow Fever Vaccine Viremia Neutralizing Ab titer Days post vaccination YFV-17D is a live attenuated vaccine. A single immunization (~10 5 pfu, subcutaneous) generates long-term immunity with neutralizing antibodies appearing by day14. Ideal model to study CD8 T cell responses to a primary, acute viral infection in humans. (Miller et al. Immunity 2008)
Yellow Fever Virus Vaccine Max Theiler receives the Nobel Prize in Physiology or Medicine from the hands of His Majesty the King Gustaf Adolf VI on December 10, 1951.
Tracking YFV-specific CD8 T cells YFV A2-NS4B 214 Tetramer %YFV Tetramer + / CD8 Day 0 0.02 Day 11 Day 14 Day 30 Day 90 2 3.6 7.5 3.3 CD8 YFV-17D genomes / ml plasma Akondy et al J.Immunol, 2009
Differentiation of YFV specific memory CD8 T cells Days post YFV Days post YFV Days post YFV Days post YFV
% α4β7+ Expression of homing receptors on YFV specific CD8 T cells % CLA+ α4β7 CLA n = 7 n = 15 11 14 30 90 Days post vaccination 11 14 30 90 α4β7 and CLA are transiently expressed by YFV specific effector CD8 T cells
α4β7 Co-expression of α4β7 and CLA on YFV specific CD8 T cells Tetramer + Total CD8 T cells Day 11 Day 14 Day 30 Donor 1 Donor 2 Donor 3 CLA
YFV memory CD8 T cells re-express CD45RA Tetramer positive cells Total CD8 T cells 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 CCR7 10 1 10 0 10 0 10 1 10 2 10 3 10 4 10 1 10 0 10 0 10 1 10 2 10 3 10 4 10 1 10 0 10 0 10 1 10 2 10 3 10 4 CD45RA Days post YFV
YFV memory cells look like terminal effectors CCR7 CCR7 10 4 T CM Naive 10 3 10 2 T EM Terminal Effectors (T EMRA ) CD45RA 10 1 10 0 10 0 10 1 10 2 10 3 10 4 CD45RA
YFV specific CD8 T cells are polyfunctional IFN Day 0 14 30 90 10 5 10 4 1.3e-3 0.1 0.6 0.2 TNF 10 3 10 2 0 IFN IL-2 10 5 10 4 0 10 3 10 2 0 0 0.09 0.07 0.3 0.3 0.1 0.06 IFN 10 4 0 0.2 0.5 0.2 Mip-1 10 3 10 2 0 IFN 10 5 10 4 10 3 0 0.2 0.5 0.2 CD107A 10 2 0 IFN 10 5 10 4 10 3 0 4.05e-3 0.09 0.08 0.04 0.55 0.005 0.17 CD45RA 10 2 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
CCR7 YFV specific effector CD8 T cells have an unique gene expression signature YF YFV 3760 170 T CM 189 YF YFV 3609 321 T EM 388 YFV 3530 400 TE 567 The number of genes that are modulated relative to naïve CD8 T cells (Fold change cutoff 1.8, p < 0.05) T CM Naive T EM TE (T EMRA ) CD45RA
Summary YFV specific CD8 T cells appear to pass through an effector phase before undergoing memory differentiation. YFV specific effector CD8 T cells transiently express homing molecules to not only the skin (CLA) but also the gut (α4β7) even though the vaccine is given subcutaneously. A window of opportunity for effector cells to migrate to the mucosal sites. Effector CD8 T cells are characterized by a gene expression signature that is distinct from that of memory and effector T cell subsets. YFV specific memory cells re-express CD45RA and continue to be CCR7 negative, a phenotype associated with terminal differentiation. However, they are polyfunctional and exhibit high proliferative potential.
Number of YFV-tetramer positive CD8 T cells / ml blood Persistence of the YFV-specific CD8 T cell response Absolute counts (Tetramer+ cells per ml of blood)
How are memory CD8 T cells maintained? Truly long-lived, quiescent cells Rapid replenishment (division and death)
Using 2 H 2 O to determine the lifespan and turnover of YFV-specific CD8 T cells Deuterium ( 2 H 2 ) is a non-radioactive isotope of hydrogen. 2 H 2 O intake is safe and has no effect on physiologic processes at the dose used. Labeling can be monitored by sampling body water from saliva, urine or plasma. GC/MS used to quantify labeled DNA is sensitive and reproducible (inter-experiment variation s.d < 0.1%). It makes in vivo analysis in humans possible. Hellerstein MK, et al. J Clin Invest, 2003
Experimental design Vaccinate HLA-A2+ donors with YFV-17D Deuterated water intake by vaccinees from days 0 to 14 (100 to 150ml per day) Sort YFV-specific tetramer + CD8 T cells at days 14, 21, 28, 42, 70.. till ~ day 365 Isolate DNA and Analyze by GC/MS
% labeled YFV-specific CD8 T cells Life-span and turnover of YFV-specific CD8 T cells 2 H 2 labeled cells can be seen more than 1 year after vaccination 2 H 2 O Days post vaccination
Conclusions Label die-away is minimal with a half life of ~500 days. YFV-specific memory CD8 T cells are dividing once every 500 days with a very long intermitotic phase. Thus, the YFV vaccine is inducing truly longlived memory CD8 T cells in humans.