(PRIMARY) IMMUNE DEFICIENCIES

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1 Neil Romberg MD UPENN/Perelman SOM Children s Hospital of Philadelphia Division of Immunology and Allergy (PRIMARY) IMMUNE DEFICIENCIES

2 immunity is nearly invisible

3 immunity is nearly invisible, unless defective

4 the jobs of clinical immunologists -identify the defect & restore immune function

5 Severe Combined Immune Deficiency (SCID) T-B-NK- (ADA) T-B-NK+ (RAG, ARTEMIS) T-B+NK- (γc, JAK3) T-B+NK+ (CD3, 22q deletion) cell-mediated defect; opportunistic infections, viruses fungus (candida, PJP) mycobacterium humoral defect; sinopulmonary infections, encapsulated bacteria

6 Early SCID dx and BMT determines survival but how can affected infants be identified? HSCT <3.5 mo Survival 96% HSCT >3.5 mo Survival 66% Buckley, NEJM 1999

7 T Cell Receptor Excision Circles (TRECs) Jennifer Puck, UCSF

8 TREC Dried Blood Spot Assay Guthrie Card 50 ul blood/drop 3 mm hole punched from blood spot ~3 ul blood Extract DNA Measure TRECs by PCR Jennifer Puck, UCSF

9 California TREC screening results, year one 500,000 TREC Screens 99.2% normal first test 99.99% normal after 1 or 2 TREC tests 30 (60%) normal after flow cytometry 0.01% (n=50) Immediate Positive or DNA Amplification Failure 20 abnormal: 1 in 25,000 births 6 Typical SCID 1 Leaky SCID, Omenn syndrome 3 Variant SCID Estimated incidence of SCID Pre TREC screen, 1:200,000 Post TREC screen, 1:50,000 4 Syndromes with low T cells 6 Secondary T lymphopenia Jennifer Puck, UCSF

10 SCID screening using TRECs is nearly universal

11 Courtesy of the Jeffrey Modell Foundation identifying PID patients without a screen is art 2 or more warning signs (kinda) predicts PID sensitivity, 56%; specificity, 16%

12 what are the barriers to MDs considering PIDs? immune defects are rare; severe ones are often sporadic infections are common in pediatrics, not just PID family history is hard (and potentially messy) maternal IgG is present for first months of life presentation is the result of a gene defect and exposure for disease, so presentation is delayed and variable there are now hundreds PID diagnosis

13 the diagnosis of PID patients is relies heavily on technology # of PIDs Million $ 1 Million $ 100K $ 10K $ 1K $ 100 $ 10 $ 1 $ 10 1 million base pairs Cost to sequence Data courtesy of NHGRI

14 the greatest teachers of modern immunology: patients with immunodeficiency diseases. Robert A. Good M.D. PhD. PIDs illuminate the cellular and molecular basis of: -immunity (candida, pneumococcus, and mycobacteria) -immune tolerance -cytotoxicity and cancer susceptibility

15 the molecular basis of candidal control at mucosal sites Image courtesy of G. Colm

16 candida covers our mucocutaneous surfaces but some have more than others

17 candida covers our mucosal surfaces but some have more than others

18 COMMENSAL CANDIDA EPITHELIUM STERILE SUB-MUCOSAL TISSUE MUCO- CUTANEOUS SURFACE

19 COMMENSAL CANDIDA EPITHELIUM STERILE SUB-MUCOSAL TISSUE MUCO- CUTANEOUS SURFACE

20 DECTIN CARD9 TLR2&4 NF-κB IL-6 IL-23 IL-1β MyD88 IRAK-1 IRAK-4 defects in candidal detection

21 defects in T 17 differentation IFNγR STAT1* 2011 DECTIN * CARD9 * T H 0 T-bet T H 1 STAT3* TLR2&4 NF-κB IL-6 IL-23 IL-1β 2009 MyD88 IRAK-1 IRAK-4 RORγC* T H

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23 Chemokines IL-17RA* IL-17RC* IL-17/IL-22 specific defects DECTIN * CARD9 * T H 0 IFNγR STAT1* T-bet T H STAT3* TLR2&4 MyD88 NF-κB IL-6 IL-23 IL-1β RORγC* 2011 IRAK-1 IRAK Β-defensins NF-κB ACT1 * IL-17* IL-22* T H 17

24 Chemokines IL-17RA* IL-17RC* DECTIN * CARD9 * T H 0 IFNγR STAT1* T-bet T H 1 STAT3* TLR2&4 NF-κB IL-6 IL-23 IL-1β RORγC* MyD88 Β-defensins NF-κB IRAK-1 IRAK-4 ACT1 * IL-17* IL-22* T H 17 A perfect candida detection and response system for your home (mucus)

25 the molecular basis of mycobacterial immunity Image courtesy of Z. Nielson

26 Q: what does pictured woman on the left have in common with the one on the right?

27 A: childhood exposure to M. bovis (BCG vaccine)

28 normal abnormal BCG vaccine BCG lymphadenitis BCGosis

29 IL-2 IL-2R IL-12R T / NK IFN IL ? IFN R IL AFB Salm. TNF NRAMP1 M TNF R CD14 LPS

30 IL-2 IL-2R IL-12R T / NK IFN IL-12 GATA2 AFB Salm. IL- 15 NRAMP1 18? STAT1 ISG15 NEMO M CD14 LPS 1 2 IRF8 TLR IFN R TNF TNF R MSMD Mendelian Susceptibility to Mycobacterial Disease

31 IL-12R IL-2 IL-12 Salmonella Coccidioides Histoplasma Burkholderia T / NK AFB Salm. IL-2R GATA2 IL- 15 NRAMP1 18? STAT1 ISG15 NEMO M CD14 IFN LPS extra-pulmonary mycobacterial 1 2 IRF8 TLR IFN R control ONLY TNF TNF R MSMD Mendelian Susceptibility to Mycobacterial Disease

32 IL-2 IL-12R IL-12 T / NK AFB Salm. IL-2R GATA2 IL- 15 NRAMP1 18? STAT1 ISG15 NEMO M CD14 IFN LPS 1 2 IRF8 TLR endogenous and pharma anti-cytokine abs IFN R TNF R TNF GM-CSF

33 anticytokine autoantibodies (Aab) An adult onset immunodeficiency In East Asia M=F, In US all F and Born SE Asia High titer Neutralizing IgG 4 90% of HIV neg Thai patients with opportunistic infections had neutralizing anti-ifng autoantibodies Labs otherwise normal, but may have elevated IgG Aab NOT in pulmonary and disseminated TB Anti-GM-CSF autoantibodies in pulmonary and meningeal cryptococcosis and nocardiosis Treatment: antibiotics/antifungals, Rituximab More to come! (likely by Steve)

34 the molecular basis of pneumococcal immunity Image courtesy of B. Gallet

35 pneumococcemia, historically a common and deadly infectious disease Figure 1: Mortality from pneumococcal bacteremia Watson et al, 1993, Clin. Infect. Dis.

36 although Th17 cells contribute to pneumococcal immunity in mice they are expendable in humans In humans pneumococcal immunity is mediated by serum soluble proteins complement deficient patients are susceptible to coccemia due to poor opsonization Courtesy of CDC

37 In humans pneumococcal immunity is primarily mediated by antibodies Courtesy of G. Kaiser

38 most PID patients with pneumococcal susceptibilities possess B-cell defects Cunningham-Rundles et al, 2005, Nat. Rev. Immunol.

39 no B cells = no antibodies 8 y.o. male with 19 episodes of pneumococcal sepsis absent gamma peak on protein electrophoresis IM IgG initiated monthly at 100mg/kg It works! B-cell lymphopenia in XLA described 2 decades later Bruton 1952, Pediatrics

40 B cells are present but germinal center responses are defective: the hyper IgM syndromes Ki67 CD20 CD20 Control Control Control CD40L deficient patient ICOS deficient patient AID deficient patient Facchetti et al. 1995, J. Immunol (Warnatz et al. 2006, Blood) (Revy et al. 2000, Cell)

41 what can we learn about B-cell tolerance mechanisms from PID patients? Cunningham-Rundles et al, 2005, Nat. Rev. Immunol.

42 B-cell tolerance occurs at distinct developmental stages bone marrow peripheral blood germinal center reaction pro-b cells early immature B cells new emigrant B cells mature naϊve B cells immune protective antibody titers health V(D)J recombination central tolerance checkpoint peripheral tolerance checkpoint terminal tolerance checkpoint non-autoreactive B cells poly-reactive B cells (Adapted from Cambier, 2007 Nat. Rev. Immuno) self-reactive B cells (Adapted from Wardemann et al., 2003, Science)

43 PID defects reveal the molecular players in central and peripheral B-cell tolerance bone marrow peripheral blood germinal center reaction pro-b cells early immature B cells new emigrant B cells mature naϊve B cells immune protective antibody titers health V(D)J recombination central tolerance checkpoint peripheral tolerance checkpoint terminal tolerance checkpoint non-autoreactive B cells poly-reactive B cells self-reactive B cells BTK CD19* MyD88 IRAK4 TACI* CTLA4* CD40L FOXP3 MHCII DOCK8 AID/UNG TACI* NFKB1* Courtesy of Eric Meffre * = CVID associated genes

44 non-infectious CVID Complications are primarily autoantibody mediated Autoantibodymediated autoimmunity ITP AIHA Evan s Syndrome Autoimmune neutropenia Pernicious anemia Myasthenia gravis Other autoimmunity Thyroiditis Autoimmune hepatitis T1DM Cunningham-Rundles et al., 2010, Blood.

45 the most bizarre germinal centers ever, CVID patients with autoimmune cytopenias (AICs) A Control CVID-AIC Y37 CVID-AIC C63 Fig. 2 CVID+AIC Y45 CVID+AIC Y40 CVID+AIC Y28 Le Coz et al, In review

46 like AID deficiency, CVID patients with AICs lack typical germinal center products IgA (mg/dl) serum IgA ** somatic hypermutations (SHM) CVID-AIC CVID+AIC FIG E2. Serum IgA concentrations are consistenly lower in CVID patients with AICs. Lower limit of adult normal IgA concentration range is indicated by a dashed line. Column heights indicate each group s mean. Statistically significant differences are indicated, **P <0.01 (Mann- Whitney U test). class-switched memory B cells usage HD CVID-AIC CVID+AIC * ** ** Le Coz et al, In review

47 somatic hypermutation mediates clonal redemption in germinal centers Apapted from Sabouri et al. 2014, PNAS)

48 Take home messages PIDs may be rare diseases but they offer key insights and proof of concept translational opportunities for astute clinicians and scientists. They are easy to miss, don t do that