The document discusses a study examining the effect of Mycobacterium avium infection on T cell differentiation in the thymus. The study found that (1) the thymi of infected mice retained the ability to generate new T cells, but (2) T cells differentiated in infected thymi had an impaired ability to protect against M. avium in peripheral organs compared to those differentiated in non-infected thymi. Specifically, T cells from infected thymi showed a reduced ability to produce IFN-gamma in response to M. avium antigens. The results suggest that infection induces central tolerance specifically to the infecting pathogen.

2. Introduction
• The host immune response against Mycobacteria
infection depends, to a great extent, on the activation of
infected cells.
• Mycobacteria-specific CD4 IFN gamma
• The thymus is a target for mycobacterial dissemination,
independent of the route of infection used

3. Hypothesis
• What extent T cell differentiation is preserved
and whether the newly generated T cells, whose
differentiation occurred within infected thymi,
differ from those generated in noninfected
thymi.

4. Methods
Animal Model:
• C57BL/6 (WT), Nude (B6.Cg-foxn1) and TCR alfa KO
• Famele
• 8-10 week-old
• 106 CFU M. Avium (2447)

5. Methods
• Thymic transplant
• TCR alfa KO mice removed (10-20min)
– DMEM and XL&KT
• Immunization
– OVA 3T at 1wk intervals (10µg s.c)
• Sacrificed 1wk after lmm

6. Methods
Measumment
General aspect of organs
Histopathology H&E
Slides were visualized using: Abs (anti-rabbit
Mycobacterium spp, K5, K8, anti CD11c or anti
Immunofluorescence Image analysis F4/80
Secondary Abs: anti-rat IgG Alexa Flour 594 or
Alexa Flour 488, anti-rabbit IgG Alexa Flour 488
and Biotinylated anti hanster IgG + PBCS
.No synthase (iNOS)
Quantification of TCR TREC Quantitaive RT-PCR using TCRA constant
RT-PCR rearrangement excision gene
circles
ELISPOT The stimuli used IFN-gama
Ag85A241-260 was quantified by ELISA
Flow cytometry cells were labeled CD3, cells were labeled with Abs specific for CD3
CD4 and CD8 (145-2C11), CD4 (RM4-5), and CD8 (53-6.7,)
Statistical analysis The two-tailed Student Differences among the means of experimental
t test. groups were analyzed with the two-tailed
(P < 0.05) Student t test

7. Results
Infected cells within the thymus are CD11c+
• Using Abs specific forK8 (subset of K8+ cell within the medulla) and K5 (subset
of K5+K8+ cell cortico-medullary region scarttered in the cortex )
• As infection progressed, bacteria were typically present within clumps of large
cells, mostly at the cortico-medullary region and within the medulla.
• All infected cell stain for CD11c consisted two populations macrophages
(CD11c+F4/80+) and DC (F4/80-)

8. Results
Representative thymic sections from M. avium-infected WT mice (20–24 wk
postinfection) stained with Abs specific for CD11c ( B–E),

9. Results
CD4- CD8- CD3-
CD4+ CD8-+CD3low/-
CD4+ CD8- CD3+
CD4- CD8+ CD3+
The mycobacterial infection did not result in altered total Only minor difference detected, in infected or Non-
thymic cell number up to 22 wk postinfection. infected after 22wk in 4 thymocyte populations
No differences were detected in the amount of TRECs up to
30 wk postinfection
These results suggest that thymocyte differentiation is maintained throughout
chronic mycobacterial infection.

10. Result
What extent the newly generated T cells,
whose differentiation occurred within infected
thymi, were able to mount a protective
immune response against mycobacterial
infections?

11. Results
Nude mice have no thymus but have competent
T cell precursors, and TCR a mice have a thymus,
but their T cell precursors are unable to fully
differentiate.

12. Results
T cells arising from infected or noninfected thymi were equally able to
colonize the periphery of nude recipient mice.
Mice transplanted with noninfected thymi presented almost ten times fewer
viable bacteria in the liver and spleen than those transplanted with infected
thymi
T cells that differentiate within infected thymi have an impaired ability to
T cells that differentiate within infected thymi have an impaired ability to
protect against the same pathogen in peripheral organs.
protect against the same pathogen in peripheral organs.

13. Results
splenocytes were stimulated in vitro
The prodution of IFN is smiliar in
WT an Non infected

14. Result
The number of IFN gamma producing
splenocytes, when stimulated with Ag85 or
with OVA , was assessed by ELISPOT. Each bar
represents the mean 6 SD of the number o f
IFN-gamma producing cells from four to five
mice per group. pp # 0. 05.
T cells that differentiate in an infected thymus have a reduced ability to
produce IFN- g in response to M. avium Ags

15. Results
Each bar represente the mean SD
from five to nine mice per group
infected thymis is have minor ability to
expressed iNOS

16. Discussion
• Thymi infected with M. avium retain the ability to generate new T
cells. However, T cells generated within infected thymi are unable
to mount a protective response against M. avium in the periphery.
• The infected and noninfected thymi (or of isolated T cells recovered
from transplanted recipients) have a similarities in the ability to
repopulate the periphery, As well a their indistinguishable ability to
produce IFN-g in response to Con A.

17. Discussion

18. Discussion
• T cells that differentiate within infected thymi mount an immune
response to OVA that is similar to that of T cells that differentiate in
noninfected thymi.
• The T cell tolerance observed against M. avium Ags in animals
transplanted with infected thymi seems specific to this pathogen.
– Inability to control bacterial growth
– Reduced aptitude to produce IFN-gamma upon stimulation (mycobacterial
Ags)
T cells are able to mount T cell-specific immune responses

19. Discussion
• Mycobacteria were detected within macrophages and DCs in
the medulla and at the cortico-medullary region .

20. Conclusions
During infection with M. avium, infected cells within the thymus induce
During infection with M. avium, infected cells within the thymus induce
tolerance specifically to mycobacterial Ags . .
tolerance specifically to mycobacterial Ags
This is the first report showing induced central tolerance to an infecting
pathogen
Knowing that the thymus is also a target organ for
mycobacterial infections in humans, it is now important to
evaluate to what extent the newly generated T cells from
patients infected with M. avium or M. tuberculosis display an
impaired ability to respond to mycobacterial Ags.

21. Thank you
For your attention!